ANALGESIC AND IMMUNOMODULATORY EFFECTS OF
CODEINE AND CODEINE 6-GLUCURONIDE
By
VINAYAK JAYA SRINIVASAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1996
i.~' ■■....
>\ ? ^ ; i -- / ACKNOWLEDGMENTS
I would like to take this opportunity to offer my heartfelt appreciation to Dr.
Ian Tebbett. His continous guidance and encouragement was the driving force
for this research project. I would like to express my sincere thanks to the other
members of my committee-Dr. Donna Wielbo, Dr. Kenneth Sloan, Dr. Hartmut
Derendorf and Dr. Roger Bertholf-for all their advice and suggestions throughout
the project.
I want to thank Dr. James Simpkins, Dr. Janet Karlix and Dr. Guenther
Hochhaus for giving me an opportunity to work in their labs in order to generate
some of my data. I also would like to thank Jim Ketcham for his invaluable help
in putting all my posters, papers and this dissertation together.
I would also like to convey my thanks to Shawn Toffolo and Becky
Frieburger for their help in my work. I also deeply appreciate the support of all
the graduate students and the other faculty members and secretaries in the
department.
I want to express my deepest gratitute to my brother, my relatives and all
my friends who have stayed by me through the thick and thin. Finally, I want to
dedicate this work to my parents because I did it for them.
TABLE OF CONTENTS
pagg
ACKNOWLEDGMENTS ii
ABSTRACT vi
CHAPTERS
1. BACKGROUND AND SIGNIFICANCE 1
1.1 Opioids and Pain 2
1.1.1 Pain Transnnission 3
1.1.2. Pain Perception 4
1.2 Codeine 4
1.2.1 Administration and Dosage 5
1.2.2 Pharmacological Actions 6
1.2.3 Toxicity 7
1.2.4 Therapeutic Uses 7
1.2.5 Drug Dependence and Tolerance 8
1.2.6 Analytical Techniques 9
1.2.7 Pharmacokinetics in Man 10
1.2.7.1 Absorption 11
1.2.7.2 Distribution 11
1.2.7.3 Metabolism 12
1.2.7.4 Elimination 14
1.2.8 Pharmacokinetics in Rats 16
1.2.8.1 Absorption 16
1.2.8.2 Distribution 17
1.2.8.3 Metabolism 18
1.2.8.4 Elimination 19
1.3 Drug Glucuronidation 20
1.3.1 Overview 20
1.3.2 Direct Pharmacological Activity 21
1.4 Evaluation of Analgesia in Small Animals 26
1.4.1 Introduction 26
III
1.4.2 Time Course of Analgesic Effect 27
1.5 Genetic Polymorphism 28
1.6 Immunomodulation 29
1.6.1 Opioid Receptors 30
1.6.2 Effects on Lymphocytes 31
1.6.3 Effects on Myleoid Cells 32
1 .6.4 Effects on Natural Killer Cells 34
1.6.5 Mechanism of Action 34
1.7 Receptor Binding 37
1.8 Hypotheses 39
1.9 Specific Objectives 39
2. METHODS 41
2.1 Specific Objective #1 : Analytical Method 41
2.1.1 Materials 41
2.1.2 Extraction Procedure 41
2.1.2.1 Human urine 41
2.1.2.2 Rat plasma 42
2.1.2.3 Rat brain 42
2.1.3 Chromatographic Conditions 43
2.1.3.1 HPLC system 1 43
2.1.3.2 HPLC system 2 44
2.2 Specific Objective #2: Synthesis of Codeine 6-glucuronide 45
2.2.1 Reaction Step I 45
2.2.1.1 Dry benzene 47
2.2.1.2 Fresh silver carbonate 47
2.2.2 Reaction Step II 48
2.2.3 Reaction Step III 48
2.3 Specific Objective #3: Analgesic Activities of Codeine and
Codeine 6-glucuronide 48
2.3.1 Intracerebroventhcular Route Studies 49
2.3.1.1 Surgery 50
2.3.1.2 Tail flick method 50
2.3.2 Subcutaneous Route Studies 51
2.3.3 Intravenous Route Studies 51
2.3.4 Statistics 52
2.4 Specific Objective #4: Immune Studies with Human
T Lymphocytes 52
2.4.1 Method 53
2.4.2 Statistics 55
2.5 Specific Objective #5: Receptor Binding Studies 55
IV
2.5.1 Materials 55
2.5.2 Method 56
2.6 Specific Objective #6: Plasma and Brain Concentrations 57
3. RESULTS 59
3.1 HPLC Development 59
3.1.1 Extraction Recoveries 61
3.1.2 Range / Linearity of Standard Curve 61
3.1.3 Specificity 62
3.1.4 Sensitivity / Limit of Detection and Quantitation 63
3.1.5 Precision and Accuracy 63
3.1.6 Stability 65
3.2 Synthesis 65
3.3 Analgesia Studies 66
3.3.1 Intracerebroventricular Route 66
3.3.2 Subcutaneous Route 70
3.3.3 Intravenous Route 83
3.4 Immune Studies 105
3.5 Receptor Binding Studies 111
3.6 Plasma and Brain Concentrations 116
4. DISCUSSION 121
REFERENCES .....:...... 132
BIOGRAPHICAL SKETCH 149
-, f
^■t
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ANALGESIC AND IMMUNOMODULATORY EFFECTS OF
CODEINE AND CODEINE 6-GLUCURONIDE
By
VINAYAK JAYA SRINIVASAN
May 1996
Chairman: Dr. Ian Ronald Tebbett
Major Department: Pharmaceutics
The interactions between opioid analgesics and the human immune
system can have important clinical consequences. A better understanding of
these interactions is needed due to the widespread use and abuse of opiates. In
recent years, an increased knowledge and awareness in this area has generated
a considerable surge in research. Narcotics are predominantly used to alleviate
pain and discomfort in patients with trauma or undergoing major surgery.
However, they are also known to cause impairment of the immune system.
Subsequently, this could lead to patients becoming predisposed to infectious
diseases as a result of the immunosuppressive effects of narcotics.
An HPLC system was successfully developed for the analysis of codeine
and its metabolites in various biological samples, that is, plasma, urine and brain
tissue. Codeine 6-glucuronide and an intermediate compound were synthesized
using a modification of the Koenigs-Knorr reaction. The synthetic procedure was
VI
efficient and reproducible. Analgesia studies with the tail flick method showed
that codeine 6-glucuronide and the intermediate exhibited a higher analgesic
activity compared to codeine when administered intracerebroventricularly.
However, both compounds were not as active as codeine when administered by
subcutaneous and intravenous routes. Immunomodulatory studies showed that
the glucuronide metabolites of codeine and morphine were less
immunosuppressive compared to their parent compounds, especially at
physiologically relevant concentrations. Receptor binding profiles of codeine 6-
glucuronide and the intermediate were similar to codeine, indicating that they
possessed activity towards the |i-opioid receptors.
The overall goal of the project was to correlate the analgesic and
immunomodulatory effects of codeine and codeine 6-glucuronide. This would
result in a better understanding of the significance of high levels of codeine 6-
glucuronide present in the plasma and urine in man after codeine administration.
Further, this may lead to the development of glucuronide analogs for the
management and treatment of pain in immunocompromised patients.
VII
CHAPTER 1
BACKGROUND AND SIGNIFICANCE
Pain is an unpleasant sensation that can disturb the comfort, thought,
sleep, and normal daily activity of a person. Pain signals are considered to be
part of a protective mechanism designed to indicate the presence of a potentially
dangerous condition. Thus, it is considered to be symptomatic of an underlying
condition that requires attention and treatment. Pain is the net effect of complex
interactions of ascending and descending neurosystems which include
biochemical, physiological, psychological, and neurocortical processes. Also,
since pain is a very subjective experience, only the patient can describe its
intensity. This subjectivity makes it difficult to assess the activity of analgesics in
humans.
Analgesics are defined as drugs that can relieve pain without causing loss
of consciousness. The most potent analgesics are referred to as narcotics and
act directly on the central nervous system. Narcotics as a group include the
opioids, which are considered to be the most effective analgesics available. The
opioid family, whose name derives from opium, includes agents such as
morphine, codeine, meperidine and methadone. While opioid is a general term
for any drug, natural or synthetic, that has actions similar to morphine, the term
opiate is more specific and applies only to compounds present in opium such as
morphine and codeine. Apart from acting as analgesics, opioids produce a
variety of pharmacological actions on various tissues in the body.
1.1 Opioids and Pain
Opioids represent the main class of drugs in the clinical management of
mild to moderate pain in various cases of medical illness, and relieve pain
primarily through direct actions on receptors in the central nervous system.
Opioid analgesics include natural alkaloids from opium (morphine, codeine),
synthetic surrogates (methadone, meperidine) and endogenous peptides
(enkephalins, p-endorphins). , .
Opioids act at receptor sites both within and outside the central nervous
system. Binding studies with various drugs and ligands in the brain and other
tissues suggest the presence of a multitude of opioid receptors. The three
important receptor types are designated as mu {\x), kappa (k) and delta (5). The
effects mediated by the |i receptors include supraspinal analgesia, respiratory
depression and euphoria. The k receptors mediate analgesia at the level of the
spinal cord, along with sedation and miosis. The 5 receptors are also thought to
be involved in analgesia, both at the spinal and supraspinal sites. However, their
role in this regard remains controversial (Jaffe and Martin, 1985).
The body produces three families of peptides that are capable of
interacting with opioid receptors-enkephalins, (3-endorphins and dynorphins.
These endogenous opioids have a high affinity for the ^, k and 5 receptors,
respectively. Ttiey are present throughout the body and serve as hormones and
neurotransmitters. It is thought that morphine and other opioid analgesics mimic
the actions of these endogenous ligands by binding to the opioid receptors.
These interactions are presumed to give rise to the observed pharmacological
effects.
1.1.1 Pain T ransmission
Pain generally begins with a noxious stimulus that injures or destroys
tissues. Endogenous chemical substances such as histamine, bradykinins,
prostaglandins, and others are then released from the damaged tissues and
nerve terminals. The released chemicals bind to "pain receptors" or nociceptors
present along the afferent nerve fibers, depolarizing the nerve membranes and
initiating an action potential. This causes the generation of a pain impulse which
is then transmitted via the afferent fibers to the spinal cord as shown in the figure
below (Figure 1-1). When the pain signals arrive at the spinal cord, they are in
turn relayed to the higher centers of the brain-thalamus and cortex.
Descending
Modulation
MIDBRAIN
Nucleus
Raptie Magnus
MEDULLA
Sp4r«o- Thalamic
Tract
Pen-AQu«^uctal Oray
Lateral Reticular
Formalion
Medial Reticular
Formation
From
Primary Afferent
Pain Fibres
Figure 1-1 : The pain modulating system (adapted from Puntillo, 1988).
»>■■■ *h
1.1.2 Pain Perception
Although some responses are reflexive in nature (e.g., knee jerk), the
perception and appraisal of pain usually occurs in the higher centers of the brain.
These systems are known to be responsible for attention, mood, motivation and
arousal. Thus, pain is perceived in the thalamic and forebrain levels and
evaluated in the cortex (Puntillo, 1988).
The perception and reaction to pain varies with each individual. It is now
evident that a host of biochemical substances, including neurotransmitters and
endogenous opioids, can modulate pain by either facilitating or inhibiting the
transmission of pain impulses at various levels of the nervous system. There is
evidence that the pain suppression system is mediated in part by endogenous
opioids along the descending pathway, which relays processed information in
response to the pain stimulus (Figure 1-1). The administration of exogenous
opioids, like morphine and codeine, is thought to enhance this pain suppression
system. However, the exact relationship between the analgesic effect of opioids
and the role of pain modulators is yet to be clearly established.
1.2 Codeine
Codeine (Figure 1-2) is a naturally occurring alkaloid in which the phenolic
hydroxy! group of morphine is replaced by a methoxy group. It was isolated in
1832 by Robiquet from the opium plant, papaver somniferum. Barbier in 1834
was the first to report its analgesic activity in humans (Baselt and Cravey, 1989).
H3C0
HO
Figure 1-2 : The structure of codeine (adapted from Muhtadi and Hassan,
1981).
Codeine is a white crystalline powder which, when made anhydrous,
melts at 154-156 °C. The phosphate salt is more soluble than the sulfate salt and
hence it is used more commonly. The free base is sparingly soluble in water, but
freely soluble in alcohol. Codeine is a monoacidic, weak base with a pKa value
of 8.2 (Baselt and Cravey, 1989). It exhibits a characteristic UV absorbance peak
in water at 284.8 nm (Grasselli and Ritchey, 1975). The anhydrous base has a
nominal molecular weight of 300.
1.2.1 Administration and Dosage
Codeine is usually given orally as a phosphate or sulfate salt for the relief
of cough and mild to moderate pain. The phosphate salt may also be given
parentally for the relief of pain by intramuscular or subcutaneous injection.
As an analgesic, the usual oral dose is 30 to 60 mg every four or six
hours, as needed for the relief of pain. For treatment of cough, the usual adult
dose is 10 to 20 mg every four to six hours, not to exceed 120 mg. As with other
opiate agonists, the smallest effective dose must be given in order to minimize
the development of tolerance and physical dependence.
1.2.2 Pharmacological Actions
Codeine, like morphine, acts by blocking excitatory synaptic transmission
in the central nervous system and relieves pain and anxiety primarily by raising
the pain threshold. It exerts a combination of depressing and stimulating effects
on the central nervous system and various peripheral organs. Important CNS
effects include analgesia, euphoria, sedation and respiratory depression.
Supression of the cough reflex is a well-recognized action of opioids, particularly
codeine. Miosis (constriction of pupils) is another pharmacological action seen
with virtually all opioid agonists. Codeine can also cause activation of the brain
stem chemoreceptor trigger zone to produce nausea and vomiting.
Peripheral effects of codeine include increasing the tone and decreasing
the rhythmic contractions of different types of smooth muscles. In the
gastrointestinal tract this produces constipation, which may be troublesome in
ordinary analgesic therapy, but useful in the treatment of diarrhea (Jaffe and
Martin, 1985). Urethral and biliary tract spasms are usually increased by
codeine, but the analgesia produced may outweigh these undesirable effects. It
can, however, be life threatening in cases of asthma when combined with
repiratory depression.
Other effects of codeine include central vasomotor capacity depression
and dilatation of some vessels, including the coronary arteries. On the whole,
these circulatory effects are small and probably result from a combination of
central actions and peripheral histamine release. Some anticholinergic activity
may be present, but is probably not critical (Way and Adier, 1962).
1.2.3 Toxicity " .'^ :■
Codeine shares the toxic potentials of the opiate agonists. The most
common side effects observed after the administration of therapeutic doses of
codeine include dizziness, sedation, nausea, vomiting, sweating and a feeling of
light-headedness. Other adverse effects that can be seen include euphoria,
dysphoria, weakness, headache, insomnia, anorexia, gastrointestinal distress,
bradycardia and even urinary retention.
Toxic effects of opioid overdose produce a classic triad of signs : coma,
respiratory depression and constricted pupils. Breathing becomes shallow and
irregular and may slow to as low as 2-4 breaths per minute. A severe overdose
of codeine can cause respiratory depression, cyanosis, extreme somnolence
which can progress to a coma, and severe hypotension with bradycardia. This
could lead to apnea, circulatory collapse, cardiac failure and finally death.
Codeine toxicity can be treated successfully with an intravenous administration
of the narcotic antagonist naloxone (Cutting, 1972 ; Jaffe and Martin, 1985 ;
McEvoy, 1990).
1 .2.4 Therapeutic Uses
Codeine is a mild analgesic indicated for symptomatic relief of moderate
pain. It is considered to be 1/10 to 1/6 as potent as morphine as an analgesic. It
is also used as an antitussive, alone or in combination with other antitussives or
8
expectorants, in the symptomatic relief of non-productive cough (Cutting, 1972 ;
Jaffe and Martin, 1985 ; McEvoy, 1990).
1.2.5 Drug Dependence and Tolerance
The major limitation of opioids is that they characteristically produce "drug
habituation" or "addiction". Drug dependence is marked by tolerance-the gradual
deveopment of resistance to the effects of the drug after repeated administration.
Tolerance is manifested by a decline in the effectiveness of a drug, requiring a
gradual increase in the dosage in order to maintain the initial effect.
Psychic dependence is a clinical term to indicate habituation. It is defined
as compulsive use of a drug by an individual who feels euphoric and a sense of
well-being from its chronic use. This kind of dependence is seen to a lesser or
greater extent with numerous agents like caffeine, nicotine, salicylates and
bromides as well as narcotic analgesics.
Physical dependence, on the other hand, deals with the biochemical and
physiological adaptation of tissues to a new chemical environment after repeated
use of a drug. The drug becomes necessary for normal tissue function and its
withdrawal causes an abnormal cellular response referred to as "abstinence
syndrome". This situation is usually characterized by effects opposite to those of
the pharmacological effects of the drug.
The phenomenon of physical dependence can actually be visualized as
being due to the prolonged occupation of the receptor sites within the cells of the
central nervous system by opioid analgesics. This receptor-drug interaction leads
to adaptive changes in the latent cellular excitability. These changes then
manifest themselves during drug abstinence as symptoms of withdrawal. The
intensity of the withdrawal syndrome is proportional to the amount and duration
of drug administration.
1.2.6 Analytical Techniques
Until recently there was little pharmacokinetic data described in the
literature regarding low doses of codeine. This was mainly due to the lack of
analytical techniques of sufficient sensitivity and specificity. Earlier studies relied
on colorimetric assays which were not very sensitive (Woods, Muehlenbeck and
Mellett, 1956). Johannesson and Woods (1964), Yeh and Woods (1969, 1970)
used high doses of radiolabeled codeine and were able to measure codeine and
biotransformed morphine in rat plasma.
As codeine undergoes extensive metabolism, forming active metabolites,
there is a need to develop an assay to precisely determine the extent of
formation of each metabolite and quantify its potential contribution to the overall
analgesia and/or toxicity associated with codeine. The older analytical methods
to determine the levels of codeine and some of its metabolites included
radioimmunoassay (Findlay et al., 1977 ; Gintzler et a!., 1976), gas
chromatography (Jain et al., 1977 ; Kogan and Chedchel, 1976) and gas
chromatography-mass spectroscopy (Ebbighausen et al., 1973 ; Cone et al.
1983) techniques.
Although radioimmunoassays offer the sensitivity required for the
detection of these compounds, differentiation between very similar species like
morphine 6-glucuronide and morphine 3-glucuronide cannot be achieved. The
ability to identify and quantify the above metabolites is important, since morphine
6-glucuronide is known to be pharmacologically active. Gas chromatography and
10
mass spectroscopy can offer both sensitivity and specificity required for
examination of codeine and morphine, but the techniques involve time-
consuming derivatization steps and are not suitable for the glucuronides. Many
researchers have turned their attention to the development of rapid, sensitive
and specific HPLC methods for the detection of opiates.
Numerous HPLC-based methods have been reported with ultraviolet
(Persson et a!., 1989), fluorescence (Chen et al., 1989 ; Tsina et al., 1982) and
electrochemical (Harris et al., 1988 ; Svensson 1986 ; Verway-van Wissen et al.,
1991 ; Besner et al., 1989 and Bedford and White, 1985) detection systems.
HPLC with fluorescence detection requires the conversion of some compounds
to fluorescent products before analysis. Electrochemical detection does not allow
simultaneous detection of all the compounds, due to differing redox potentials.
HPLC with ultraviolet detection would therefore seem to be the method of choice
for developing an assay for all of the compounds of interest.
A potential problem with using a reversed phase HPLC system is that the
polar glucuronides elute very close to the solvent front and are prone to being
hidden by co-eluting endogenous substances (Chari et al., 1991). An alternate
method has been described for morphine and its metabolites using a normal
phase system (Wielbo et al., 1993).
1.2.7 Pharm acokinetics in Man
There is an abundance of literature describing the pharmacokinetics of
codeine in man (Quiding et al., 1986 ; Guay et al., 1987 ; Chen et al., 1991 ; Yue
et al., 1989 a , 1990 a, b ; Shah and Mason, 1990 a ; Way and Adier, 1962 ;
Findlay et al., 1977, 1978, 1986 ; Hull et al., 1982 ; Rogers et al., 1982 ; Persson
11
et al., 1992 ; Vree and Verway-van Wissen, 1992). Despite this extensive
documentation, the relevance of some of the active metabolites of codeine is not
clear. The extent of formation of individual metabolites and their potential
contributions to the analgesic efficacy seen after codeine administration need to
be assessed in detail.
1.2.7.1 Absorption
Codeine is well absorbed following oral and intramuscular administration
in man. Its bioavailability after oral administration was found to be 50-60%
(Rogers et al., 1982). At a dose of 60 mg, a peak plasma concentration (Cmax)
of around 100-200 ng/ml was seen within one to two hours after administration
(Mohammed et al., 1993). Chen et al. (1991) observed that the mean tmax (tifne
at which the peak plasma concentration is observed) for codeine occurred about
1 hour after oral administration in the case of both single and chronic dosing.
However, the mean peak plasma concentration after chronic dosing was
significantly higher than after single dosing. This was also observed by Quiding
et al. (1986) and can in part explain why some subjects experience a greater
analgesic effect after chronic dosing compared to single dosing.
1.2.7.2 Distribution
After the drug is absorbed into the blood, it is distributed to tissues in the
body. However, only the free drug concentration can equilibrate with these
tissues. The main interaction in blood is between the plasma proteins and the
drug molecules and this is usually a reversible physical process. Thus, the
binding of drugs to plasma proteins is a dynamic process. Codeine has been
shown to be bound to plasma proteins to an extent of 25-30% (Findlay et a!.,
1977). Baselt and Cravey (1989) reported a volume of distribution of about
12
3.5 I/kg for codeine, indicating an extensive distribution in the various tissues of
the body.
1 ■2.7.3 Metabolism
Codeine is primarily metabolized in the liver, the major site being the
microsomes in the endoplasmic reticulum. Lesser, but significant sites include
the central nervous system, kidney, lung and placenta. Metabolism is
predominantly via conjugation with glucuronic acid at the 6-position (Yue et al.,
1989 a, b, 1990 a, b ; Chen et al., 1991 ; Vree and Venway-van Wissen, 1992).
Other metabolic pathways include 0-demethylation to morphine and N-
demethylation to norcodeine (Sindrup et al., 1990). These primary metabolites of
codeine are further metabolized to their glucuronides as outlined in Figure 1-3.
The hepatic biotransformation of codeine to morphine has led many to
believe that that codeine may exert its analgesic effect through partial conversion
to morphine (Adier and Latham, 1950 ; Findlay et al., 1978 ; Yue et al., 1990 a).
This assumption is supported by the low affinity of codeine to the [i opiate
receptor and by the marked in vivo analgesic efficacy of morphine, morphine-6-
glucuronide and normorphine (Lasagna and Kornfeld, 1958 ; Osborne et al.,
1988). However, studies by Quiding et al. (1986) and Shah and Mason (1990 a)
have questioned the possible role of morphine in codeine analgesia because of
the very low concentrations of morphine seen after both single and repeated
doses of codeine.
Chen et al. (1991) described codeine 6-glucuronide pharmacokinetics in
detail after single and chronic oral codeine administration. There was no
difference in the plasma tmsx (1.28 versus 1.13 h) and Cmax (1 43 versus 1.38
(ig/ml) for single and chronic dosing, respectively. The mean AUCs for codeine
13
and codeine 6-glucuronide at steady state were not significantly different for
either single or chronic dosing. The average ratio of the AUCc6G • AUCcod was
Codeine-6-giucuronide
Glucuronidation
NCH3
CH3O ' o
0-demethyIation / Codeine
_NCH3
N-demethylation
NH
Glucuronidation
OH .
M3G, M6G Morphine
-«. N-demethylation
CH30 \ ■
Norcodeine
O-demethylation
Normorphine
Figure 1-3 : The various metabolic pathways of codeine.
about 15 for both single and chronic dosing regimens. Vree and Verway-van
Wissen (1992) reported similar values for the various pharmacokinetic
parameters of codeine 6-glucuronide except in the case of t-1/2 and AUC ratio.
14
While they reported ti/2 values for codeine and codeine 6-glucuronide as 1.5
and 2.8 hours respectively, Chen's group found the t-1/2 values for both
compounds to be similar, about 3 hours. Vree's group reported a AUCcsG •
AUCcod '"3^'° °^ "10 compared with 15 by Chen's group.
Quiding et al. (1986) reported plasma concentrations of morphine both
after single and multiple doses of 60 mg of codeine to be about 2-3% of that of
codeine. Shah and Mason (1990 a) found that Morphine AUG values ranged
from 2-5% after a 60 mg oral dose of codeine. However, Vree and Verway-van
Wissen (1992) reported that no free morphine could be detected in the plasma of
human volunteers who took 30 mg of codeine orally. Any morphine formed was
immediately glucuronidated at the 3- and 6- positions to form the corresponding
glucuronides. Both glucuronides were detected in the plasma with the morphine
3-glucuronide concentrations being higher than those of morphine 6-glucuronide.
Very small amounts of normorphine, norcodeine and its glucuronide conjugate,
norcodeine 6-glucuronide, were also detected in the plasma.
1.2.7.4 Elimination
Codeine and its metabolites are excreted almost exclusively by the
kidneys. A very small fraction is eliminated as free codeine (about 5%). The
major portion of the administered dose appearing in the urine consists of
biotransformed products. Urinary recoveries of codeine and its metabolites
indicate that codeine 6-glucuronide is the major metabolite formed from codeine.
There are also trace amounts of morphine and its glucuronides along with
normorphine and its glucuronide conjugate (Chen et al., 1991). The excretion of
codeine and its metabolites in 24 hour urine as a% of the dose of administered
codeine is summarized in Table 1-1. Adier et al. (1955) observed that, after a
- - r • '
f^-V ..... . t
15
single dose of codeine, urinary excretion was almost complete in 24 hours,
although trace amounts of codeine and morphine stayed in the body for several
days before being completely eliminated. A small percentage of the dose (0.02-
0.17%), consisting mostly of free codeine and some metabolites, was also
detected in the feces.
Codeine / Metabolites
% Excreted in 24 Hour Urine
Codeine
8-16
Morphine
0.5-1
Codeine 6-glucuronide
48-69
Morphine 6-glucuronide
0.5-2
Morphine 3-glucuronide
5-8
Norcodeine
2-10
Table 1-1 : The urinary excretion data in humans after a 60 mg oral dose
of codeine, (from Yue et al., 1990 a).
Codeine has an elimination half-life of about 2-3 hours (Findlay et a!.,
1977, 1978 ; Quiding et al., 1986 ; Yue et al., 1990 a, b). Chen et al. (1991)
reported that elimination half-lives for both codeine (3.2 versus 2.9 h) and
codeine 6-glucuronide (3.2 vs 3.3 h) after both single and chronic dosing,
respectively, were not significantly different. The renal clearance of codeine is
between 67 and 265 ml/min. The creatinine clearance values of codeine in
healthy volunteers has been reported to be in the range of 90 to 132 ml/min.,
indicating that in addition to glomerular filtration, codeine can undergo active
secretion into the lumen of the proximal tubules (Chen et al., 1991).
16
1.2.8 Pharmacokinetics in Rats
The physiological disposition of codeine in various experimental animals
after relatively high doses has been extensively studied. Studies done in male
rats have shown that about half the dose (55%) of codeine undergoes O-
demethylation (Yeh and Woods, 1969). Morphine formed by the metabolic
conversion of codeine has been found in the plasma, urine, bile and feces
(Johannesson and Shou, 1963 ; Johannesson and Woods, 1964 ; Yoshimura et
al., 1970). Morphine has also been determined to be present in the brain of rats
following large doses of codeine (Dahlstrom and Paalzow, 1976).
Traditionally, the major routes of administration in rats have been
subcutaneous (s.c.) and intraperitoneal (i.p.) injections. Numerous
pharmacological experiments with rats to determine analgesic activity of codeine,
morphine (Johannesson and Shou, 1963 ; Yeh and Woods, 1969 , 1970 ; Oguri
et al., 1990) and the 3- and 6-glucuronide metabolites (Yoshimura et al., 1973 ;
Shimomura et al., 1971 ; Abbott and Palmour, 1988 ; Sullivan et al., 1989 ; Gong
et al., 1991 ; Paul et al., 1989 ; Pasternak et al., 1987 ; Smith et al., 1990), have
utilized both s.c. and i.p. routes of administration. There are a few reports in the
literature regarding intravenous administration of codeine and morphine in rats
(Dahlstrom and Paalzow, 1976 ; Shah and Mason, 1991 ; Bhargava and Villar,
1992 ; Thurston et al., 1993). Shah and Mason (1990 b) also administered
codeine orally in rats as well as by i.v. injection and compared the two routes of
administration.
1.2.8.1 Absorption
Shah and Mason (1990 b) described codeine pharmacokinetics after an
oral dose of 5 mg/kg in rats. A solution of codeine phosphate was made by
17
dissolving it in 2-4 ml of physiological saline. The drug was then carefully
delivered via gastric intubation to the fasting animals. Codeine was rapidly
absorbed after the 5 mg/kg oral dose. The mean peak plasma concentrations
were 101.3 ± 42.4 ng/ml around 6.4 + 4.5 min after dosing. After 4 hours, no
codeine could be detected in the whole blood with the HPLC method used in the
study. A large intersubject variation was observed in the absorption of codeine.
This variation may be partly explained by factors such as gastric motility and
intestinal transit time.
The mean bioavailability was calculated from oral /\L/C; * Dj ^ / i.v. AUC^
* Doral 3S 0.08 ± 0.03, that is., only 8% of the ingested dose of codeine reaches
the systemic circulation. Absorption of opiates in general is thought to occur by
passive diffusion rather than by processes invoving energy expenditure
(Christensen et al., 1984). Incomplete absorption was not considered to be a
major factor as the amounts of free codeine found in the feces were negligible
following oral administration.
1. 2.8.2 Distribution
Miller and Elliot (1955) conducted several distribution experiments in the
rat. They showed that codeine was well distributed, and capable of leaving the
the blood and concentrating in parenchymatous tissues such as liver, kidney,
lung, adrenal glands and brain. This is reflected in the large volume of
distribution of the terminal portion (Vd area ~ 5-1 I/kg), obtained from the
concentration-time profile after intravenous codeine administration.
Codeine given i.v. appears to fit a two compartment body model with a
rapid distribution and a short terminal elimination phase. This indicates that
codeine is rapidly metabolized and /or excreted. Elimination rate from the central
18
compartment (0.0569 min"'') is more than the terminal rate constant (P), due to
distribution of codeine into the peripheral compartment. The mean ratio of 0.92
for K-|2 / K21 (ratio of the inetrcompartmental rate constants) shows
approximately an equivalent distribution of codeine between the central and
peripheral compartments. Linear pharmacokinetics was exhibited by codeine at
i.v. doses of 1-4 mg/kg (Shah and Mason, 1990 b).
1.2.8.3 Metabolism
There are a few studies in the literature that examine the metabolism of
codeine in rats. These were done mostly in the 1960s and were conducted with
radioactive codeine. Plasma concentrations after a 2 mg/kg s.c. injection of
codeine showed the presence of free codeine, free morphine and conjugated
morphine (in the ratio of 10 : 3 : 1). No conjugated codeine was seen either in
the plasma or brain (Yeh and Woods, 1969). The conjugated morphine was later
characterized as morphine 3-glucuronide. It was reaffirmed that morphine 6-
glucuronide and codeine 6-glucuronide were not formed in rats (Yeh and Woods,
1970). However, using thin layer chromatography, a Japanese group
(Yoshimura, 1970) detected traces of codeine 6-glucuronide (0.2%) in the urine
of rats, showing that a small amount of this metabolite can be formed in rats.
This has been supported by Oguri et al. (1990) who used a specific HPLC
method and found the urinary recovery of this compound to be about 1% (Table
1-2). Recent findings (Oguri et al., 1990 ; Lawrence et al., 1992) have shown that
morphine 6-glucuronide could not be detected in plasma or urine of rats after
codeine administration. . ■ .. .. , .
-f . 4; ' >> ^
19
1. 2.8.4 Elimination
In vivo codeine disposition studies using radiolabeled carbon-14 have
shown that about 74% of the injected radioactivity is excreted as free codeine,
free morphine and morphine conjugate via the pulmonary, biliary, intestinal and
urinary routes in male rats (Yeh and Woods, 1969). Of this 74%, 20-40% is
eliminated in the expired air as CO2 via the pulmonary pathway.
Codeine / Metabolites
% Excreted in 24 Hour Urine
Codeine
1 -2
Morphine
4-5
Codeine 6-glucuronide
0.1-0.3 1
Morphine 6-glucuronide
Not detected
Morphine 3-glucuronide
23-24
1 Norcodeine
Not detected
Table 1-2 : Urinary excretion data as a% of a dose of codeine (from
Ogurietal., 1990).
A significant part of an i.v. dose of codeine has been shown to undergo
enterohepatic recirculation to the extent of 10-30% (Walsh and Levine, 1975). It
is known that codeine can decrease gastrointestinal tract motility, thereby
increasing transit time of morphine glucuronide in the intestinal tract. This would
then allow longer exposure to bacterial glucuronide hydrolysis which, in turn,
would favor enterohepatic recycling. Yeh and Woods (1969), using radioactively-
labeled tracers, reported the following amounts recovered from intact bile : free
codeine (1.3%), free morphine (0.9%) and conjugated morphine (43.1%).
20
Oguri et al. (1990) saw a substantial interspecies difference in the
metabolism of codeine (Table 1-3). This can influence considerably the nature
and duration of the pharmacological and toxicological activities of codeine. The
development of molecular aspects of gene evolution has been applied
extensively to explain species differences seen in drug metabolism (Nebert and
Gonzalez, 1987).
Codeine/ IVIetabolites
Mouse
(n=25)
Rat
(n=4)
Guinea Pig
(n=4)
Rabbit
(n=3)
Codeine
6.8 + 0.7
1.6 + 0.2
1.6 + 0.2
2.2 + 0.5
Morphine
0.8 + 0.2
4.3 + 0.4
0.2 + 0.1
1.3 + 0.3
Codeine 6-glucuronide
1.6 + 0.2
0.2 + 0.1
39.8 + 3.9
24.5 + 3.7
Morphine 6-glucuronide
nd
nd
0.7 + 0.1
1.9 + 0.3
Morphine 3-glucuronide
7.6+ 1.0
23.9 + 2.8
1.6 + 0.2
17.9 + 1.4
Norcodeine
9.0 + 0.8
nd
nd
nd
nd = not detected
Table 1-3 : Urinary excretion data (% of dose) 24 hours after codeine
administration in various species (from Oguri et al., 1990).
1.3 Drug Glucuronidation
1.3.1 Overview
A major pathway for drug metabolism and excretion is the generation of
water soluble glucuronide metabolites. Since many drugs exhibit structural
features that allow conjugation without previous phase I reactions,
glucuronidations are viewed as first line detoxification mechanisms. Most
21
glucuronides of drugs are considered to be inactive and rapidly eliminated.
Therefore, glucuronide nnetabolites of drugs are often neglected in
pharmacodynamic and pharmacokinetic studies and are not taken into account
when evaluating drug effects.
The pharmacological and toxicological relevance of glucuronidation was
pioneered by Dutton and is summarized in a comprehensive review by Kroemer
and Klotz (1992). The general reaction scheme which describes the conjugation
of glucuronic acid to various drugs in the presense of glucuronosyl transferase
enzymes is shown in Figure 1-4. This reaction mediates the formation of ether,
ester, thiolic, N- and C- glucuronides.
Uridine diphosphate glucuronosyltransferases (UGTs) are enzymes
located in the endoplasmic reticulum and therefore form a part of the microsomal
fraction. UGTs are generally 50 to 60 kD in size and span the entire membrane
of the endoplasmic reticulum. There is a small C-terminal domain located in the
cytoplasm with the active site directed toward the lumen of the endoplasmic
reticulum.
1.3.2 Direct Pharmacological Activitv
The best documented example of glucuronide activity is that of morphine.
Morphine is conjugated to give morphine 3-glucuronide and morphine 6-
glucuronide in the liver (Wahlstrom et al., 1988 ; Coughtrie et al., 1989).
Shimomura et al. (1971) observed that morphine 6-glucuronide had a direct
analgesic effect in the hot plate test. Subsequently, the same group
demonstrated that both morphine 6-glucuronide and morphine 3-glucuronide can
penetrate the blood brain barrier (Yoshimura et al., 1973). Using the tail flick
22
"° I <\0 - (P) ®- OH2C s. /O.
GA
OH OH
UOP
UOP gfucuronosyl
transferase
COOH
+ UDP
Figure 1-4 : Glucuronidation of a substrate (H-S) by reaction with
uridine diphosphate-glucuronic acid (UDP-GA) in the presence of UDP-
glucuronosyl transferase enzymes (from Kroemer and Klotz, 1992).
latency tests in rats, they found that morphine 6-glucuronide was 20-fold more
potent than morphine after direct microinjection into the periaquaductal gray area
of the brain. Morphine 3-glucuronide in this experimental design did not produce
any effect.
The observations of Pasternak have been confirmed by a number of
investigators (Paul et al., 1989), who performed detailed characterization of
morphine 6-glucuronide. After peripheral administration to rats the analgesic
effect was twice that of morphine itself. After intrathecal administration, morphine
6-glucuronide was reported to be 650 times more potent than the parent
compound. Smith et al. (1990) showed that morphine 3-glucuronide had no
analgesic activity but could act as a potent antagonist of morphine and morphine
6-glucuronide induced analgesia in rats. In this context Woolf (1981) reported
that morphine 3-glucuronide was capable of inducing hyperalgesia in rats.
23
Recent investigations of force field and quatum mechanical characterization of
morphine 3-glucuronide and morphine 6-glucuronide reveal an unexpectedly
high degree of lipophllicity (Carrupt et al., 1991).
The question which then arises is whether this detailed pharmacological
evidence for the contribution of glucuronides to the net drug effects of morphine
is matched by clinical observations. Joel et al. (1985) speculated that morphine
6-glucuronide may contribute to the clinical efficacy of morphine. Hanks et al.
(1987) tried to explain the potency of repeated oral doses of morphine as due to
accumulation of morphine 6-glucuronide. Direct clinical evidence for the
analgesic action of morphine 6-glucuronide was obtained by Osborne et al.
(1988), who injected morphine 6-glucuronide, 1.0 mg/kg to 5 patients and 0.5
mg/kg to 1 patient. Five patients reported total pain relief within 30 minutes and
the analgesia lasted for 1-7 hours. Pain and pain relief were monitored by visual
analog scales (Figure 1-5). :
Pharmacokinetic Parameters
Patient A
(Normal Renal
Function)
Patient B 11
(Impaired Renal
Function)
Clearance (I/kg) **
89
26 II
Volume of Distribution (1)
14.7
16.4
Elimination Half-life (h) **
1.9
7.4
1 Area Under the Curve (nmol r'h) **
370
1319
indicates that the parameters were significantly different.
Table 1-4 : Pharmacokinetic parameters of two patients after a 1 mg/kg
intravenous dose of morphine 6-glucuronide (from Osborne et al., 1988).
24
1 00 T
^ 80
u
E
3
g 60
'x
CO
E
o
40
20
TIME (hours)
Figure 1-5 : A visual analog scale used for monitoring the extent of pain
relief (from Osborne et al., 1988).
250 T
4 6
TIME (hours)
Figure 1-6 : The pharmacokinetic profile of two patients after 1 mg/kg i.v.
administration of morphine 6-glucuronide (from Osborne et al., 1988).
25
Pharmacokinetic indices for two patients are shown in Table 1-4. Patient
A had normal renal function while patient B had chronic renal impairment. The
elimination of morphine 6-glucuronide was closely related to renal function. No
morphine or morphine 3-glucuronide levels were detected in the plasma at any
time. Plasma morphine 6-glucuronide levels for the two patients are shown in
Figure 1-6.
Hannah et al. (1990) investigated the analgesic efficacy of intrathecal
morphine 6-glucuronide in comparison with morphine in 3 patients with chronic
cancer pain. The doses required for controlled analgesia were 393 + 227 mg/24
h and and 227 ± 114 mg/24 h for morphine and morphine 6-glucuronide
administration, respectively.
Conjugation of drugs by glucuronosyl transferases plays an important role
in the overall picture of drug disposition. The resulting glucuronides represent
metabolites that are not always inactive and may in fact contribute to drug action
either directly (by producing analgesia as in the case of morphine 6-glucuronide)
or indirectly (by release of the parent compound via hydrolysis as in
enterohepatic recycling). Moreover, some glucuronic acid conjugates are not
rapidly excreted. Their disposition can be modulated at different levels of
distribution, metabolism and excretion, thereby modifying net drug action.
Therefore, glucuronic acid conjugates should be taken into account when
pharmacokinetic and pharmacodynamic characterization of drugs are
determined.
26
1.4 Evaluation of Analgesia in Small Animals
1.4.1 Introduction
Eddy (1928, 1932) is credited as being the first to describe methods for
determining analgesia in animal experiments by exerting a variable pressure on
the distal part of the cat tail. Friend and Harris (1948) used a pair of forceps,
whereby pressure could be exerted on the tail of the rat. Green et al. (1951)
produced pain by exerting pressure on the tip of the tail using a syringe piston
system.
Macht and Macht (1940) were the first to describe a method in which
electrical stimulation was used to produce pain in rats. They implanted two
electrodes in the skin of the scrotum. The rats reacted with a squeak response
when the voltage was increased over a certain threshold. Luckner and Magun
(1951) implanted two electrodes in the upper part of the tail. Collins et al. (1964)
implanted an electrode in the rectum and another in the upper part of the tail in
rats.
Eddy et al. (1950) placed mice in a cylindrical glass container, the base of
which was a copper plate. This plate was maintained at a temperature of 50-55
°C by a hot water bath placed below the copper plate. The mice responded to
the heat by licking their forepaws and trying to jump out of the cylinder. This
technique is called Eddy's hot plate method and is a good pain model for the rat
and mouse. D'Amour and Smith (1941) irradiated the tail tip with heat from a light
source of defined strength. This tail flick method is also widely used for
determinating analgesia in small animals.
27
A modification of the tail flick method was described by Berglund and
Simpklns (1988). It involves measurement of the withdrawal time of the tail when
a beam of light was focused on it. This was done both before drug
administration, and at regular intervals thereafter. The former measurement was
called baseline latency and the latter test latency. The Instrument used was a
model 33 tail flick analgesia meter (lite Inc., Landing, NJ, USA), and consisted of
an incandescent light bulb with the beam intensity and sensitivity dial set at 75
and 8, respectively. The time between presentation of a focused beam of light
and removal of the tail was recorded as the latency period. However, in the
absence of any response, a cut-off period of 40 seconds was used to prevent
tissue damage. This was considered as the maximal suppression of pain.
1.4.2 Time Course of Analgesic Effect
D'Amour and Smith (1941) were the first to determine the analgesic effect
of morphine and codeine administered by i.p. injection. They found the effect to
be maximal at 30 minutes post-injection for both drugs. Ercoli and Lewis (1945)
injected equianalgesic doses of morphine and codeine, both intraperitoneally
and subcutaneously. They found that both drugs had their greatest effect 30-60
minutes after administration, with the effect persisting for 60-120 minutes.
Following higher doses, the degree of analgesia was found to be more complete.
Miller and Elliott (1955) were the first researchers to make a serious
attempt to look into changes in the amounts of codeine and morphine in the
brain with time. These investigators administered 25 mg/kg codeine-N-I^CH by
subcutaneous injection to rats. These rats were killed after 15, 30, 60 and 150
minutes respectively. The spinal cord and the brain were removed, and the
28
concentrations of these drugs were determined in the spinal cord, hypothalamus,
cerebellum, medulla oblongata, mid-brain and parts of the hemispheres.
Miller and Elliott (1955) reported that concentrations of codeine rose
sharply from the 15th to the 30th minute, while a slower rise was seen from the
30th to the 60th minute. The highest concentration of codeine was measured 60
minutes after drug administration. Another important fact to be considered is that
relatively small amounts of both codeine and morphine enter the brain, and it is
these concentrations which are responsible for producing analgesia. These
results indicate a relationship between drug concentrations of codeine and
morphine in the brain and the degree of analgesia produced. The exact
relationship is yet to be firmly established. < ' *
1.5 Genetic Polymorphism
Genetic variation is an important cause of the large differences seen in
drug metabolism between individuals. A number of isoenzymes in the
cytochrome P450 family are involved in the oxidative metabolism of several
essential drugs (Nebert et al., 1987). The entire population can be divided into
extensive and poor hydroxylators based on the extent to which they metabolize
certain drugs like mephenytoin, debrisoquine, sparteine and procainamide. It is
well known that 0-demethylation of codeine, leading to the formation of the
analgesically active metabolites morphine and morphine-6-glucuronide, is
catalyzed by cytochrome P450 IID6 isoenzyme (Nebert et al., 1989) and
cosegregates with the debrisoquine/sparteine oxidative polymorphism .
Five to ten percent of Caucasians are classified as "poor metabolizers"
(PMs), since they lack the specific isoenzyme for 0-demethylation (Yue et al.,
29
1990 a, b), while the remainder, capable of 0-demethylating codeine, are termed
"extensive metabolizers" (EMs). PMs are rare in the native Chinese population
compared to Caucasians (Yue et al., 1989 a) indicating inter-ethnic differences in
drug metabolism. This pharmacogenetic bias translates clinically into the fact
that the PMs may not derive as much analgesic effect as the EMs. There are no
data in the literature regarding genetic factors influencing the N-demethylation of
codeine.
Glucuronidation reactions are catalyzed by a group of isoenzymes with
overlapping specificities. Glucuronidation of codeine has been shown to be
induced by smoking and oral contraceptives (Yue et al., 1990 c). It is also
affected by the co-administration of other drugs like diazepam and
chloramphenicol. ; f; f ■'. j ; : ? ^ ' '-''"'- ' \
1.6 Immunomodulation
The immune process consists of a number of concerted events which
include recognition and processing of foreign antigens, proliferation and
differentiation of responder cells, and the production of proteins and peptides for
the amplification and mediation of the immune response. In this regard, the
immune system is not isolated and autonomous in nature. In fact it is a part of an
interactive and communicative triad, that also includes the nervous and
endocrine systems.
The immune system can affect brain functions as evidenced by the
release of neurotransmitters and enhanced brain activity in certain localized
regions after activation of the immune system following bacterial infection
(Saphier, 1987). A number of centrally acting pharmacological agents have been
30
shown to affect immune function directly or indirectly via the hypothalamo-
pituitary-adrenal (HPA) axis. Within this axis, feedback loops have been
identified which, when activated by certain immune products such as cytokines,
are responsible for the production of glucucorticoids, leading to marked
immunomodulatory effects. These effects are enhanced in stressful situations
(Herz, 1993). This is further indicative of the relationship between the brain and
the immune system, either by neuronal pathways or by modulation of endocrine
system
Drugs of abuse, particularly opioids, can produce deleterious effects on
the immune system. From an immunological standpoint, it is necessary to
determine whether the effects of drug abuse were due to the drugs, or the
consequence of needle sharing and poor nutrition--both of which are frequently
observed factors associated with drug addiction. Earlier epidemiological findings
suggested that increased infections were caused by sharing of unsterilized and
contaminated needles. However, subsequent clinical studies have focused on
the increasing evidence that the opioids themselves are capable of affecting the
host defense mechanisms by directly acting on the immune system.
1.6.1 Opioids Receptors
The concept of opioids as immunomodulators is based on studies
showing the presence of classical opioid receptors on the surface of cells of the
immune system. Ovadia et al. (1989) demonstrated that rat lymphocytic
membranes possess a certain GTP binding protein that couples opioid receptors
to adenylate cyclase in response to the action of an opioid on the lymphocytes.
Wybran et al. (1979) reported an opioid involvement in immune function based
31
on their observations with human T lymphocytes. They indicated that the opioid-
induced suppression of lymphocytes was reversible with naloxone.
Carr et al. (1994) found that naltrexone antagonized both the analgesic
and immunosuppressive effects of mice, suggesting the involvement of the same
receptor for both actions, that is, the |i receptor. This inference is supported by
the findings of Ward et al. (1984) which showed that a ^-selective antagonist |3-
funaltrexamine, and not the 5-selective antagonist naltrindole (Portoghese et al.,
1988) or the K-selective antagonist nor-binaltorphimine (Portoghese et al., 1987),
attenuated morphine-induced immune suppression.
1.6.2 Effects on Lymphocytes
Lymphocytes are the primary immunocytes responsible for cell-mediated
(T lymphocytes) as well as humoral (B lymphocytes) immunity as seen in Figure
1-7. Measurement of the effect of various drugs on the proliferative capacity of
lymphocytes is the most common in vitro assessment of functional cell-mediated
responses. A large number of lymphocyte markers are suppressed following
acute and/or chronic administration of opioids (Herz, 1993).
Naloxone-reversible reduction in the number of circulating lymphocytes in
morphine-treated rabbits has been reported along with effects on the various
subpopulations of lymphocytes (Herz, 1993). Arora et al. (1990) found an
increase in the T helper to T suppressor ratio following morphine treatment. T
lymphocyte rosette formation was one of the first functional measures of T ceil
function to be studied for comparing the immunosuppressive potential of various
drugs. Wybran et al. (1979) showed that the suppressive effects of opioid
agonists, in particular morphine, on the T cell rosette formation was
32
stereospecific for levorotatory forms, naloxone reversible, and produced
tolerance.
Humoral immunity, on the other hand, involves antibody responses to
drugs which are recognized as antigens by the immune system. Primary
antibody responses were seen to be suppressed by the action of opioids in
sheep erythrocytes (Lefkowitz and Chiang, 1975). Plaque-forming responses,
which are indicative of antibody production, were suppressed in splenocytes
obtained from mice implanted with morphine pellets (Bryant et al., 1990).
1.6.3 Effects on Myeloid Cells
Cells of myeloid origin include monocytes, macrophages, neutrophils,
mast cells, basophils and eosinophils. In addition to serving as mediators of
inflammatory responses, they are involved in numerous functions critical to early
immune response. These functions include antigen presentation, antibody
production, lysis of tumor cells, phagocytosis of foreign particles and the release
of immune response mediators such as interferons, cytokines, transforming
growth factor (TGF) and tumor necrosis factor (TNF).
In humans, morphine exposure leads to a depression of the phagocytic
properties associated with myeloid cells (Tubaro et al., 1985). Macrophages,
which are activated by the stimulatory agent y-interferon, were inhibited in
morphine-pelleted mice and the tumoricidal activity of the macrophages was
seen to be completely abolished (Bryant et al., 1988 a). Chronic morphine
administration in mice also inhibited macrophage colony formation as the result
of a decreased expression of the macrophage stimulating factor (Herz, 1993).
33
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34
1 .6.4 Effects on Natural Killer Cells
Natural killer cells represent another type of immune cell and constitute
only about 5% of the total leukocyte population. They are large cells possessing
an intrinsic activity for non-specific killing and lysis of a variety of tumor cells. In
addition to serving as scavengers of malignant cells, natural killer cells also play
a regulatory role in antibody production.
Shavit et al. (1984) demonstrated that daily doses of subcutaneous
morphine (50 mg/kg) suppressed natural killer cell tumoricidal activity (NK
activity), and that naltrexone administration abolished this effect. This is strong
evidence that the natural killer cytotoxic effect is centrally-mediated. Shavit et al.
(1986 a) injected small quantities of morphine directly into the lateral ventricles
and found that the NK activity was markedly suppressed. Novick et al. (1989)
reported a marked decrease in NK activity in heroin addicts on methadone
maintenance therapy. ' '
NK activity is usually below normal basal levels in HIV patients and
declines as the disease progresses. Endogenous opiate peptides, leucine-
enkephalin and methionine-enkephalin have been reported to increase the
cytolytic capacity of the natural killer cells (Wybran et al., 1987 ; Oleson and
Johnson, 1989). This indicates that such candidates can potentiate the NK cell
responses and restore immunocompetence in the case of pre-AIDS and AIDS
patients.
1.6.5 Mechanism of Action
Opioids have been shown to down-regulate immune responses. The
exact mechanism by which this immunosuppression occurs has yet to be
35
established. A direct mechanism of action is thought to be through lymphocyte
opioid receptors (Figure 1-8). The second hypothesis is that the immune effects
are indirectly mediated, either by the activation of the hypothaiamic-adrenal-
pituitary (HPA) axis with subsequent increase in the production of adrenal
corticosteriods or by the release of catecholamines as a result of sympathetic
innervation.
It has been observed that systemic administration of morphine
suppresses the activity of NK cells in the rat (Shavit et al., 1984, 1986 a, b). The
same group also reported that central administration of morphine produced the
similar results, but required doses one third of those administered systemically.
The NK suppression was blocked by naltrexone. N-Methyl morphine, a morphine
analog which cannot cross the blood brain barrier, had no effect on the NK
cytotoxicity when administered systemically. This is further evidence that the
immunosuppressive effects of morphine are mediated by opioid receptors in the
brain.
Morphine and other opioids are known activators of the HPA axis, and
induce glucocorticoid output. Corticosterone has been implicated as possessing
potential immunosuppressive effects as it was able to dose-dependently
suppress NK activity in vitro. This effect was also observed in vivo in mice
implanted with morphine pellets (Freier and Fuchs, 1994). A glucocorticoid
receptor antagonist, RU 38486, blocked morphine-induced suppression of NK
activity in a dose-dependent fashion. Naltrexone administration antagonized the
morphine-induced elevation in serum corticosterone. This suggests that
suppression of NK activity is linked to glucocorticoid elevation, which is the result
3&
MORPHINE
O
mMUNE SySTEM: boTs^
marrow, lymph nodes, thymus,
spleen, somatic tissues, vascular
space
Figure 1-8 : The mechanisms of opioid-lnduced immunosuppression (from
Peterson eta!., 1990).
studies have shown that exposure of immune cells to opiates, especially
morphine, results in a variety of functional disturbances (Chao et al., 1992, 1993;
Peterson etal., 1991,1993).
There are many reports in the literature implicating opiates as
immunomodulatory agents. Most of these studies have focused on morphine, the
narcotic of choice In case of severe pain associated with trauma and cancer, and
have shown that morphine possesses potent immunosuppressant activity. There
are, however, no studies that investigate the immunosuppressive effects of
codeine or any of the glucuronide metabolites of codeine and morphine.
A technique that simulates cellular immune response in vitro is the mixed
lymphocyte reaction (MLR). It is based on the observation that lymphocytes from
a mixture of genetically different individuals with different HLA (Human
Leukocyte Antigen) types react with each other and proliferate. This test is
performed by preventing the response of one set of lymphocytes (donor) through
37
radiation which then allows only the other set of lymphocytes (recipient) to
proliferate as shown in Figure 1-9 .
in
in
0)
c
c
>,
W
<
Z
Q
I I I I I I
12 3 4 5 6
Days
Figure 1-9 : A mixed lymphocyte reaction with cell proliferation.
Thus, opioids possess receptors which are capable of modifying immune
functions. The concentrations achieved with analgesic doses of opioids are
similar to those reported in the in vitro immunomodulatory experiments. It is also
apparent that stress-induced activation of the endogenous opioid networks can
contribute to various immunological changes. The focus for future research must
include an understanding of the role of opioids in regulating immunity and their
interaction with other immune function mediators.
1.7 Receptor Binding
In the case of morphinans, the aromatic ring and the basic nitrogen atom
are necessary for analgesic activity. Substitutions at the phenolic hydroxyl group
(position 3) and the alcoholic hydroxyl group (position 6) have been shown to
cause pharmacological profiles which are opposite in nature. While additions at
m
the 6-position were seen to enhance opioid receptor binding affinities, changes
at the 3-position significantly decreased receptor binding (Labella et a!., 1979).
The structure activity relationships of morphine and its 3- and 6-glucuronide
nnetabolites have been evaluated in several studies (Yaksh et al., 1986 ; Gong
eta!., 1991 ; Shimomura et al., 1971 ; Pasternak et al., 1987).
On the basis of these results, investigators speculated that the inactivity of
morphine 3-glucuronide compared with morphine 6-giucuronide could be due to
the differences in the level of receptor binding. Christensen and Jorgensen
(1987) showed that morphine 6-glucuronide, but not morphine 3-glucuronide,
had a high affinity for the opiate receptors isolated from bovine brains in
competition with ^H-naioxone. Subsequent investigation by Pasternak et al.
(1987) identified that morphine 6-glucuronide interacts with [i- but not k-
receptors.
The greater potency of morphine 6-glucuronide compared to morphine in
antinociception studies has been reported by various investigators (Abbott and
Palmour, 1988 ; Sullivan et al., 1989 ; Paul et al., 1989). In contrast to morphine
and morphine 6-glucuronide analgesia, morphine 3-glucuronlde produces
hyperalgesia, respiratory stimulation and behavioral excitation by non-opioid
mechanisms (Yaksh et al., 1986 ; Pelligrino et al., 1989). These studies suggest
that morphine 3-glucuronide can actually antagonize the effects of both
morphine and morphine 6-glucuronide. This is clinically important not only for the
analgesic effect, but also for the respiratory depression associated with morphine
administration.
39
1.8 Hypotheses
The hypotheses of this project are based on the overall aim of the project,
that is, to examine and compare the analgesic and immunomodulatory effects of
codeine and codeine 6-glucuronide.
1. Codeine 6-glucuronide, like morphine 6-glucuronide, possesses
analgesic activity.
2. The glucuronide metabolites of codeine and morphine are less
immunosuppressive than their parent compounds.
1.9 Specific Objectives
1. Develop and validate a reliable and sensitive HPLC-UV based assay
for the quantitation of codeine, morphine, codeine 6-glucuronide,
morphine 6-glucuronide and morphine 3-glucuronide in biological
samples. -•. - , . ■. •■■■ .
2. Chemically synthesize codeine 6-glucuronide utilizing a modification
of the Koenigs-Knorr reaction. '^
3. Assess the immunomodulatory effects of codeine, morphine and their
6-glucuronide metabolites in human T lymphocytes (in vitro).
4. Compare the analgesic potencies of codeine and codeine 6-glucuronide
in the rat using the tail flick method after intracerebroventricular (i.c.v.),
subcutaneous (s.q.) and intravenous (i.v.) routes of administration.
5. Determine the [i opioid receptor binding affinities of codeine and
codeine 6-glucuronide.
6. Analyze plasma and brain concentrations of codeine and their
40
metabolites at the peak analgesic response time after administration of
codeine and codeine 6-glucuronide by various routes as described in
specific objective #3.
^ . *
CHAPTER 2
METHODS
2.1 Specific Objective #1 :
Analytical Method
An isocratic HPLC method was developed using a ultraviolet absorbance
detector along with an efficient solid phase extraction method to analyze
physiological concentrations of codeine, codeine 6-glucuronide, morphine,
morphine 6-glucuronide and morphine 3-glucuronide in various biological
samples, that is, human urine, rat plasma and rat brain. The method was first
developed using blank human urine. It was then validated using rat plasma and
brain samples.
2.1.1 Materials
HPLC grade methanol and acetonitrile were used (Fischer Scientific,
Fairlawn, NJ, USA). Analytical grade potassium dihydrogen phosphate and 85%
v/v o-phosphoric acid were supplied by Sigma (St. Louis, MO, USA) as were
codeine, morphine, morphine 6-glucuronide and morphine 3-glucuronide. A
sample of codeine 6-glucuronide was donated by the National Institute on Drug
Abuse (NIDA, Rockville, MD, USA).
2.1.2 Extraction Procedure
2.1.2.1 Human urine
Solid phase extraction was performed with 3 ml Clean Screen® columns
(Worldwide Monitoring, Horsham, CA, USA) containing 40 micron bonded silica
41
42
particles. The cartridges were placed on a 12 station Vac-Elut (Varian, Harbor
City, CA, USA). The columns were conditioned with methanol (3 ml), distilled
water (3 ml) and 0.025 M phosphate buffer pH 3 (1 ml). A 1 ml sample of urine
mixed with 2 ml of phosphate buffer pH 3 was then loaded onto the column. The
cartridges were air-dried for 30 seconds and then washed with 0.025 M
phosphate buffer pH 3 (1 ml), followed by methanol (1 ml). The columns were
air-dried again for 30 seconds before eluting the compounds with 3 ml of 5%
freshly prepared ammoniacal methanol solution. The eluent was evaporated to
dryness under a stream of nitrogen gas and the residue was reconstituted in 150
)Ltl of the mobile phase and 50 ^\ was injected into the HPLC system.
2.1.2.2 Rat plasma
Extractions were performed with 10 ml Clean Screen® columns. The
columns were conditioned with methanol (10 ml), distilled water (10 ml) and
0.025 M phosphate buffer pH 3 (2 ml). A 200 ^L sample of rat plasma was mixed
with 400 nL of 0.025 M phosphate buffer pH 3 and loaded onto the column. The
cartridges were air-dried for 30 seconds and then washed with 0.05 M acetate
buffer pH 4.5 (2 ml), followed by methanol (2 ml). The columns were air-dried for
30 seconds before eluting the compounds with 3 ml of 10% freshly prepared
ammoniacal methanol solution. The eluent was evaporated to dryness under a
stream of nitrogen gas. The residue was reconstituted in 150 ^\ of the mobile
phase and 50 ^1 was injected into the HPLC system.
2.1.2.3 Rat brain
Brain samples were accurately weighed and an aliquot of 0.025 M
phosphate buffer pH 3 (1 ml) was added to each sample. The samples were then
homogenized and transferred to borosilicate tubes. After addition of a further 2
m
ml 0.025 M phosphate buffer pH 3, the samples were placed in a shaker for 10
minutes. The samples were then centrifuged at 4000 rpm for 20 minutes. The
supernatant was removed and loaded onto 10 ml Clean Screen® columns. The
columns were conditioned with methanol (10 ml), distilled water (10 ml) and
0.025 M phosphate buffer pH 3 (2 ml). After the samples were loaded, the
cartridges were air-dried for 30 seconds and washed with 0.01 M acetate buffer
pH 4.5 (2 ml) followed by methanol (2 ml). The columns were air-dried for 30
seconds before eluting the compounds with 3 ml of 10% freshly prepared
ammoniacal methanol solution. The eluent was evaporated to dryness under a
stream of nitrogen gas. The residue was reconstituted in 150 ]x\ of the mobile
phase and 50 \i\ was injected into the HPLC system.
2.1.3 Chromatographic Conditions
2.1.3.1 HPLC system 1
This system was used to examine human urine samples for the presence
of opiates. It consisted of a Waters 501 multi-solvent pump set at a flow rate of
0.9 ml/min. The mobile phase consisted of 82% acetonitrile and 18% phosphate
buffer (0.05 M potassium dihydrogen phosphate adjusted to a final pH of 3 with
85% v/v o-phosphoric acid). Separation of the compounds was achieved on a 20
cm X 4.5 mm I.D. Accubond® diol column with a 5 micron particle size (J & W
Scientific Inc., Folsam, CA, USA). A Spectra-Physics Focus® multiwavelength
forward optical scanning detector (San Jose, CA) set at 220, 230 and 280 nm
was used to detect eluting compounds. Data acquisition and analysis was
performed with Autolab® software loaded on a 386 IBM computer.
44
Apart from chromatographic analysis, the Autolab software provided the
option to examine the UV spectra of the compounds in the chromatograms. All of
the compounds of interest exhibited a maxima in their UV spectra around 285
nm. A specific opiate of interest could be further characterized using derivative
spectroscopy. In derivative spectroscopy, the first or higher derivative of
absorbance with respect to wavelength is recorded versus wavelength. In this
way the ability to detect and measure minor spectral features is considerably
enhanced (Willard, 1986).
2.1.3.2 HPLC system 2
This system was used to determine concentrations of codeine, morphine
and their glucuronides in rat plasma and brain samples. It consisted of a Waters
501 multi-solvent pump set at a flow rate of 0.7 ml/min. The mobile phase
consisted of 88% acetonitrile and 12% phosphate buffer (0.05 M potassium
dihydrogen phosphate adjusted to a final pH of 3 with 85% v/v o-phosphoric
acid). Separation of the compounds was achieved on a 20 cm x 4.5 mm I.D.
Accubond® diol column with a 5 micron particle size (J & W Scientific Inc.,
Folsam, CA, USA). Sample injection was automated by the use of a Waters™
717 Plus autosampler. A Waters 486 tunable absorbance detector set at 220 nm
was used to detect eluting compounds. A Millenium® 2010 Chromatography
Manager software was used to acquire data. All the instruments were controlled
by a 386 NEC Powermate computer, which also stored and processed the
acquired chromatographic data. V'
45
2.2 Specific Objective #2:
Synthesis of Codeine 6-glucuronide
Codeine 6-glucuronide is not available commercially. In order to evaluate
its pharmacological activity in the rat, relatively large amounts of this compound
(100-200 mg) were required. The National Institute of Drug Abuse (NIDA) is the
only agency which provides samples of this compound, basically for analytical
purposes (5 mg). For this reason, it was decided to synthesize codeine 6-
glucuronide using a reproducible method described in the literature (Yoshimura
et al., 1968). The initial attempt to synthesize codeine 6-glucuronide with some
modifications of the Koenigs-Knorr reaction provided a good yield of the product,
comparable to that reported in literature. A scheme of the synthetic route (Figure
2-1) is shown below.
2.2.1 Reaction Step I
In the first step, codeine monohydrate (1 g) was dissolved in 200 ml of dry
benzene in a three-necked flask attached to a condenser with a drying tube
containing drierite (CaS04) and a Dean-Starke trap. The trap was used to
periodically distill benzene and to maintain anhydrous conditions in the flask. The
solution was heated at 150 °C with an oil bath. Small amounts (250 mg) of
freshly prepared silver carbonate along with equal portions of a solution
containing 5 g of the acetobromo derivative of glucuronic acid, that is, methyl
2,3,4-tri-0-acetyl-1-bromo-1-deoxy-D-glucupyranuronate (obtained from NBS
Biologicals Inc., Herts, UK), in 100 ml of dry benzene were added every hour
over two seven hour periods.
46
MeOOC_Q Br
^i^ (Protected Glucuronic Acid)
~^ " //
Silver Carbonate ^
\ in Benzene Me
Codeine
)Ac
"Intermediate"
Sodium Methoxide
in Methanol
Me
1 . Barium Hydroxide
2. Oxalic Acid
Codeine 6-glucuronide
Figure 2-1 : Synthetic route of codeine-6-glucuronide using the Koenigs-Knorr
reaction (adapted from Yoshimura et a!., 1968).
Thin layer chromatography (TLC) was performed at regular intervals
during the heating period to monitor the extent of the reaction. The solvent used
for the TLC was a mixture of methanol and methylene chloride (1:4). Small
portions (1 ^1) were removed from the boiling mixture and spotted on TLC plates
(Brinkman Polygram Sil G/UV 254). With increasing time, the spot representing
the starting material codeine, disappeared and another spot with a higher Rf
value appeared. This indicated the formation of a product assumed to be methyl
[codein-6-yl 2,3,4-tri-0-acetyl-1-bromo-1-deoxy-D-glucupyranosiduronate, that is,
the 6-glucuronide of codeine with intact acetyl and methyl groups on the
glucuronic acid moiety, and subsequently referred to as the intermediate. The
47
structure and identity of the compound in CDCI3 was confirmed by H NMR
(Varian EM 390 Spectrometer).
After the heating was stopped, the contents of the flask were filtered and
the clear filtrate was evaporated to dryness with a rotory evaporator. The residue
was redissolved in absolute ethanol and evaporated to dryness. The solid
residue was then transferred to a column and chromatographed using 500 ml
each of ethyl acetate, ethyl acetate : ethanol (60 : 40), ethanol : methanol (50 :
50) and methanol. Various fractions were collected and concentrated to dryness.
The dried fractions which indicated the presence of the compound of interest
were recrystallized using methanol. The melting point of the compound was 113-
116°C, in agreement with the value previously reported (Yoshimura et a!., 1968)
2.2.1.1 Dry benzene
A key point to guarantee the success of the first step was to ensure that
the benzene used was absolutely dry. This was achieved by boiling benzene in
the same apparatus as the reaction was done and removing the water with the
Dean-Starke trap. Boiling for about 4 hours ensured removal of all the water
present in the benzene.
2.2.1.2 Fresh silver carbonate
Freshly prepared silver carbonate is another essential prerequisite in the
first step of the reaction, as the silver carbonate used as a catalyst is susceptible
to oxidation in the presence of moisture and light. The procedure for preparing
"active" silver carbonate (Wolfrom and Lineback, 1963) involves adding an
aqueous solution of anhydrous sodium carbonate (1.6 g in 7.5 ml of distilled
water) dropwise into a mechanically stirred solution of 8 g of silver nitrate
dissolved in 20 ml of water. A solution of 1 g of anhydrous sodium hydrogen
48
carbonate in 12.5 ml of water was then added to the above in 2-3 portions. The
mixture foams and a yellowish precipitate is formed. The solid recovered by
filtration was silver carbonate.
2.2.2 Reaction Step II
The intermediate compound from step I (0.6 g) was suspended in a test
tube with 3.5 ml of absolute methanol. A 1% solution of sodium methoxide in
methanol (2 ml) was added and the mixture was stirred with a magnetic stirrer.
The solution was evaporated to dryness in vacuo.
2.2.3 Reaction Step III
The dried residue from step II was dissolved in 2.2 ml of a 0.43N Ba(0H)2
solution and stirred for about 4 hours before leaving it overnight in a refrigerator
at 4° C. The barium salt which precipitated on cooling was dissolved in 4 ml of
distilled water and adjusted to pH 6 with 2N oxalic acid solution. The solution
was refrigerated overnight and the barium oxalate formed was removed by
filtration. The filtrate was evaporated to dryness and the residue was
recrystallized from methanol. The compound decomposed at 225-230 °C, which
was slightly lower than that reported in the reference paper.
2.3 Specific Objective #3:
Analgesic Activities of Codeine and Codeine 6-glucuronide
The polar glucuronide metabolites of codeine and morphine do not cross
the blood brain barrier (BBB) as efficiently as their parent compounds. With this
in mind, initial studies were designed to bypass the BBB and determine if
48
codeine 6-glucuronicle produced analgesia in pain-induced rats by using
standard antinociceptive tests, that is, tail flick and hot plate methods. This was
done by delivering the compounds directly Into the brain of rats via the
intracerebroventricular route (i.c.v.). The next step was to compare the activities
of codeine and codeine 6-glucuronide after subcutaneous and intravenous
administrations using the same methods as in the i.c.v. studies for measuring the
effects.
During the synthetic procedure, the intermediate compound formed after
the first reaction step was isolated and characterized. This intermediate is the
glucuronic acid moiety attached to the 6-position of codeine with intact
acetyl/methyl groups. The antinociceptive activity of this intermediate was also
investigated.
2.3.1. Intracerebroventricular Route Studies
These studies involved investigation of the antinociceptive effects of
codeine, codeine 6-glucuronide and the Intermediate compared to the standard
analgesic drug, morphine. All drugs were dissolved In physiological saline (pH
4.5 - 5.5) and administered at doses of 100 [jg/5 pi for codeine, 10 pg/5 pi for
codeine 6-glucuronide and the intermediate, and a dose of 5 pg/5 pi for
morphine. Each compound was administered to groups of 6 rats. One group of
rats was injected with saline only and served as a control group. Another group
was not Injected with anything to control for any possible effects of the surgical
procedure on the pharmacodynamic measurements. Measurements were made
at the following time points after administration of the compounds : 0, 10, 20, 30,
40, 60, 90, 120, 150 and 180 minutes.
so
2.3.1 .1 Surgery
After the animals were acquired, they were housed in cages in a room
with a 12 hour light-dark cycle. The animals were fed on standard laboratory rat
chow and tap water ad libitum. Each rat was anesthetized with 30-50 mg/kg
sodium pentobarbital intraperitoneally and stereotaxically fitted with a 23 gauge
intracerebroventricular stainless steel guide cannula. The co-ordinates used for
the stereotaxic apparatus were : 1.0 mm lateral; 1.0 mm caudal to the bregma
and 5.0 mm below the skull surface (Paxinos and Watson, 1986). The rats were
allowed to recover for 3-5 days prior to starting the antinociceptive experiments.
2.3.1.2 Tail flick method
Analgesia was determined using the tail flick method of Berglund and
Simpkins (1988) and previously described in section 1.4. Reduction in pain was
expressed as the% of the maximum possible effect (% MPE) calculated as :
o/o MPE = ( ^QSt latency - baseline latency ) , ^^^
( cut-off period - baseline latency )
The area under the effect curve (AUEC) was determined from individual%
MPE versus time graphs using trapezoidal calculation. The AUEC is considered
a good indicator for comparing the intensity of effect during a certain time period.
It also provides a good estimate of the duration of effect produced by each
compound. Comparison of total AUEC values are, therefore, considered to
reflect the relative effectiveness of the compounds.
51
2.3.2 Subcutaneous Route Studies
After the intracerebroventricular route, studies were performed by injecting
the test compounds subcutaneously, that is, under the nape of the neck.
Codeine, codeine 6-glucuronide and the intermediate were dissolved in
physiological saline (pH 4.5 - 5.5) and injected at a dose of 10 mg/kg. A higher
dose of 20 mg/kg of codeine was also administered to a group of 6 rats. The
volume of injection was 1 ml/kg body weight of the rat. Each rat was also
administered saline in a separate study and therefore, acted as its own control.
Assessment of the reduction in pain was determined by the tail flick
method as described for the intracerebroventricular route. The intervals between
time points for measuring the response were, however, greater than in the
intracerebroventricular studies so as to take the absorption factor into
consideration. The measurement time points were 0, 15, 30, 45, 60, 90, 120,
180, 240, 300 and 360 minutes.
2.3.3 Intravenous Route Studies
In these studies, the jugular vein of rats was catheterized so that
compounds could be directly injected into the general blood circulation. Animals
were first anesthetized with ketamine/xylazine (45:9 mg/100 g body weight of rat)
intraperitoneally. A heparinized (100 U/ml) catheter (PE50 tubing, 0.58 mm X
0.965 mm) was then placed in the right jugular vein. The catheters were
stoppered and exteriorized between the scapulae to avoid chewing. The rats
were allowed 1-2 days to recover from the anesthesia and surgery. Drugs were
dissolved in physiological saline (pH 4.5 - 5.5) and injected through the catheter.
Codeine, codeine 6-glucuronide and the intermediate were administered at a
., "■»
52
.... /'\
dose of 10 mg/kg. The injection volume was 1 mg/kg body weight of the rat.
Physiological saline (500 |il) was also injected to ensure that the drug reached
the systemic circulation and did not remain in the dead volume of the catheter.
As in the intracerebroventricular and subcutaneous studies, the tail flick
method was used to determine the analgesic effect. As there was no absorption
phenomenon to consider, periods between measurement times were less than in
the subcutaneous studies but greater than the intracerebroventricular studies,
that is, 0, 10, 20, 30, 40, 60, 90, 120, 180, 240 and 300 minutes.
2.3.4 Statistics
The area under the effect curve data for each compound was compared
to its saline treatment using a paired Student's t-test. The area under the effect
curve (AUEC) of compounds were also compared with each other using an
unpaired Student's t-test.
2.4 Specific Objective #4:
Immune Studies with Human T Lymphocytes
In vitro immune studies on the drugs were performed with human T
lymphocytes found in peripheral blood mononuclear cells (PBMCs) which were
isolated from the blood of healthy volunteers using the Ficoll-Hypaque density
gradient centrifugation procedure. The isolated T lymphocytes were then
stimulated by mitogens phytohemagglutin (PHA) and phorbol 12-myristate-13-
acetate (PMA), activating the resting T cells and enabling them to become
transformed into lymphoblast cells. These transformed cells are then capable of
synthesizing DNA, dividing rapidly and proliferating.
ii-
53
When the lymphocytes reached their peak proliferation, they were labeled
with 1 pCi ^H thymidine after a period of 48 hours (for the PHA/PMA assay) and
120 hours (for the mixed lymphocyte reaction assay). The cells incorporated the
radiolabeled thymidine into their DNA and counts were done using a scintillation
counter. The differences in cell count determined with and without drugs was
used as a measure of drug-induced changes in lymphocyte proliferation.
Immunosuppression produced by drugs was expressed in terms of% inhibition of
proliferation and calculated as : .
„. . uux- rr^ ir x- (CPM wjthout clf UQ - CPM wJth df uq) ^ ^ „-
% Inhibition of Proliferation = -^^ ^ * 1 00
(CPM without drug)
2.4.1 Method
-' i ;■■■'
6 "<
Peripheral blood (20 ml) was obtained from a healthy volunteer and
transferred to a conical tube. To this conical tube 20 ml of RPMI tissue culture
media containing 5% albumin was added (this culture media was developed at
Rosewell Park Memorial Institute and hence the name, RPMI). A 10 ml Ficoll-
Hypaque solution was drawn up in a pipette and carefully delivered to the bottom
of the tube. The tube was then placed in a balanced centrifuge for 30 minutes at
1200 rpm. A white layer containing lymphocytes formed between the culture
media and red cells. The lymphocyte cells were transferred into another conical
tube and diluted with 50 ml of the RPMI cell culture media. The tube was
centrifuged again for 10 minutes at 1200 rpm. A "pellet" of cells was seen to be
formed at the bottom of the tube. The supernatant was decanted until 1 ml of the
54
media remained in the tube with the pellet. The tube was then slightly agitated to
disperse the pellet homogeneously in the media and a cell-count was done to
determine the total number of cells/ml using tryphan blue stain under a light
microscope (Fischer Scientific, Fairlawn, NJ, USA) at a magnification of 40 X.
The cell suspension was diluted with RPMI media to obtain a cell-count of 1x 10^
cells/ml for PMA and PHA assays and 2x10^ cells/ml for the MLR assay.
Stock solutions and dilutions of the drug to be tested were made in RPMI
cell culture media. A 96-well U-bottom cell plate was used and 100 pi drug
solutions of each concentration of each drug was pipetted into the plate in
triplicate. To each drug-containing well was added, 100 pi of either PHA (5
pg/ml) or PMA (50 ng/ml) containing T lymphocytes. The controls in the assay
consisted of T cells without the drug, T cells containing the mitogens (PHA or
PMA) but without the drug, and a control with only the cell culture media. The cell
plate was covered on the top and placed in an incubator until it was radiolabeled.
Standard drug dilutions were done in the same way as in the case of
PHA/PMA assays. Lymphocytes from two separate individuals were used for this
assay. The cells of one individual was selected to be irradiated and labeled as
137
the donor. This was done in a cell irradiator with a Cs source at 2500 rads for
4 minutes. To 100 pi of drug solution of varying concentrations, 50 pi of the
irradiated cells and 50 pi of the plain cells from the other individual were added.
The cell plate was covered and incubated until it was radiolabeled.
The cell plates in the PHA/PMA assay (on day 2) and MLR assay (day 5)
were taken out of the incubator and placed on the work surface. ^H-Thymidine (1
pCi) diluted with the RPMI culture media was taken out of the refrigerator. Using
55
a Hamilton microsyringe stored in ethanol, each cell well was given 10 |j| of the
^H-thymidine solution. . -
Cells were harvested onto nitromethylcellulose paper via a cell harvester
attached to a vacuum pump. The cell plate was discarded in a radioactive waste
box. Scintillation vials were set up and labeled appropriately. After the paper had
dried, forceps were used to punch out individual circles (each representing a cell
well) which were placed into the corresponding scintillation vials. Scintillation
fluid (3 ml) was then added to each vial and the vials were capped tightly.
Radioactivity counts were determined using a scintillation counter.
2.4.2 Statistics
The% inhibition of proliferation data of the compounds were compared to
each other using an unpaired Student's t-test.
2.5 Specific Objective #5:
Receptor Binding Studies
2.5.1 Materials
Tris-HCI, bovine serum albumin (BSA), morphine sulfate and naloxone
HCI were obtained from Sigma Chemicals (St. Louis, MO, USA). Sodium chloride
was purchased from Fischer Scientific Inc. (Fairlawn, NJ, USA). Codeine
phosphate was obtained from Westlab Pharmacy, Gainesville, PL. Codeine 6-
glucuronide and the intermediate were synthesized using the Keonigs-Knorr
reaction. ^H-DAGO ([ D-ala^ N-methyl-phe^ glyol^ ][ tyrosyl-3,5-^H ] enkephalin)
was used as a competitive ligand for the binding studies and was purchased
from Amersham (Arlington Heights, IL).
2.5.2 Method
Receptor binding studies were carried out using brain tissues from
Sprague-Dawley rats (200-300 g) using a modification of the procedure reported
by Hochhaus et al. (1988). Brain tissue was homogenized in 60 volumes of 50
mM Tris-HC! buffer (pH 7.4) containing 100 mM sodium chloride. The
homogenate was incubated in a water bath for 1 hour at 20 °C. After incubation,
the homogenate was centrifuged at 20,000 rpm for 20 minutes at 4 °C. The pellet
formed at the bottom was washed twice with 50 mM Ths-HCI buffer. The
membrane suspension was vortexed for 2 minutes to ensure homogeneity.
Competitive binding studies were performed after adding 1% w/v of
bovine serum albumin (BSA) to the membrane suspensions. The suspension
(880 ^l) was placed in polyethylene tubes and incubated with 20 |il of the tracer
(1 nM ^H-DAGO, a |i-receptor selective agonist) and 100 |al of the competing
ligand (various concentrations of the drugs dissolved in 50 mM TrIs-HCI buffer).
This mixture was incubated in a water bath for 1 hour at 20 °C. After incubation,
the bound and unbound fractions were separated by filtration using Whatman
GF/C filters. The filter paper containing the retained radioactivity was transferred
to scintillation vials and 3 ml of scintillation fluid was added to each vial. The vials
were capped tightly and shaken to enable the radioactivity to be distributed in the
scintillation cocktail. The vials were left overnight and tritium counts were
performed using a liquid scintillation counter.
Specific binding of morphine, codeine, codeine 6-glucuronide and the
intermediate to the )a-receptor was determined by competitive displacement of
the radiolabeled tracer by various concentrations of the test compounds. The
non-specific binding (NS) was determined with a relatively high concentration of
57
morphine (1 |iM). The total binding (T) was determined from vials in which no
drug was added. The counts per minute (CPMs) obtained from a scintillation
counter were then plotted versus increasing drug concentrations. The data was
fitted using an E^ax model from the Scientist program (Micromath Scientific Inc.,
Salt Lake City, UT) as described in Equation 1. The raw data (CPMs) for each
compound were standardized by transforming them into% of total binding and
plotting them as a function of increasing drug concentration (Equation 2). The
equations used for fitting the data are as follows :
Equation 1 :
CPM = T - ^ + NS
Equation 2
CPM
% Total Binding = * 100
2.6 Specific Objective #6:
Plasma and Brain Concentrations
The objective of this study was to determine plasma and brain
concentrations at the peak analgesic response time in the rat. This was achieved
98
by the administration of codeine or codeine 6-glucuronide to groups of rats (n=6)
by intracerebroventricular, subcutaneous and intravenous routes (described in
section 2.3). The peak response time from the tail flick experiments after each
route of administration was used as the time at which plasma and brain samples
were collected.
Rats were first anesthetized with metofane 5 minutes prior to the peak
response time. At the appropriate time, the rats were decapitated using a
guillotine. Trunk blood samples were collected in vacutainers containing EDTA
as an anticoagulant. Plasma was obtained by centrifuging the blood at 2500 rpm
for 20 minutes. Brain samples were obtained after removing the skull and other
membranes attached to the brain. Both the plasma and brain samples were
stored in a freezer at -80 °C. The samples were analyzed using the HPLC
method described in section 2.1.
'•> r
U .J
r-J. Tv;- ■>■
CHAPTER 3
RESULTS
3.1 HPLC Development
An HPLC-UV based method was successfully developed for the analysis
of codeine, morphine, codeine 6-glucuronide, morphine 6-glucuronide and
morphine 3- glucuronide in biological samples, that is, human urine, rat plasma
and rat brain. A typical chromatogram obtained after injection of a standard
solution containing 100 ng/ml of each compound is represented in Figure 3-1
(system 1). This system uses a multiwavelength scanning UV detector and
allows the simultaneous examination of the same chromatogram at different
wavelengths in the scanning range (Figure 3-2).
0.0100 1
0.0080 -
a 0.0060
I
g>
5 0.0039 -
<
0.0019
-0.0001
M
JU
C60
MCO
\ I =T ^T
8.00 11.20 14.40 17.60
■nme(min)
KOO
-I —n-
20.80
^'
24.00
Figure 3-1 : A standard solution containing 100 ng/ml of each of all the
compounds using system 1 .
59
0.0030 -,
.| 0.0024 -I
J 0.0018 •
S 0.0012
0.0006 -
0.0000
8.00
20.80
24.00
Time ( min)
Figure 3-2 : The same chromatogram as in Figure 3-1 showing the
multiwavelength capabilities of the detector.
The software also allows examination of the UV spectra at any time point
in the acquired chromatogram (Figure 3-3). This can be used to identify the
presence of an opiate, since all opiates exhibit a UV maxima of 285 nm. The
differential UV spectra further helps to confirm the identity an opiate of interest
(Figure 3-4). This is done by following minute changes in the spectra of an opiate
and matching it with the spectra obtained from a standard solution of the same
opiate. '
0.1000
0.0800
C 0.0600
3
WO
u.
15.527
<
0.0400
0.0200
0.0000
240
280
320
Wavelength (X)
Figure 3-3 : A typical UV spectra which is exhibited by all opiates.
61
N )
15.527 15.527"
Wavelength ( X )
Figure 3-4 : A normal (— ) and second derivative (— ) spectra of morphine.
3.1.1 Extraction Recoveries
Extraction recoveries for eacii compound were determined by comparing
the peak area of an extracted standard to an unextracted standard. The elution
solvent of methylene chloride-isopropanol-ammonium hydroxide recommended
by Worldwide Monitohng for opiates gave good recoveries for codeine and
morphine only, but not for the glucuronide metabolites. However, the use of a
5% ammonium hydroxide solution in methanol enabled the efficiency of
extraction of polar glucuronides from human urine samples to be increased. This
elution solvent gave clean extracts and excellent recoveries in excess of 80% for
all the compounds of interest. The% extraction recoveries for the various
compounds of interest in human urine are represented in Table 3-1.
3.1 .2 Ranee / Linearity of Standard Curve
After the initial development of the chromatographic system and extraction
procedure, calibration curves were prepared in drug-free human plasma and
62
urine. Standards were set up for each compound of interest in the biological
matrix (plasma/urine) at concentrations of 0, 10, 50, 100, 200, 400 and 500
ng/ml. Each point on the calibration curve was taken as the average of two
determinations. The standard curve was determined from calibrators by the
linear least squares fit to the equation y = mx + b, where x = concentration, y =
drug peak area, m = slope of the line and b = y-intercept of the line. Linear
correlations between the area under the peak and concentration of each
compound was performed by regression analysis using the Microsoft Excel
program. It was seen that each compound of interest had a good correlation with
an R^ of 0.99 or better over the range 0-500 ng/ml (Figures 3-5 and 3-6).
Compound
% Extraction recovery ± sd (n=4)
Codeine
92 ±6
Morphine
90 ±8
Codeine 6-glucuronide
86 ±4
Morphine 6-glucuronide
85 ±8
Morphine 3-glucuronide
83 ±6
Table 3-1 :% extraction recoveries for compounds of interest in human
urine.
3.1.3 Specificitv
Drug-free plasma and urine samples were analyzed with and without the
drugs. This was performed to show that the drug peaks were well separated from
63
the solvent front and did not have any interference from endogenous materials
also eluting from the column.
3.1.4 Sensitivity/Limit of Detection a nd Quantitation
The detection limit was determined as the concentration of the sample
which corresponds to the signal that is twice that of the baseline noise. When
extracted from human urine using Clean Screen® columns, the minimum
quantifiable concentrations of the vanous compounds using system 1 were : 5
ng/ml for codeine and morphine; 10 ng/ml for codeine 6-glucuronide, morphine
6-glucuronide and morphine 3-glucuronide.
3.1.5 Precision and Accuracv
Precision is defined as a measure of the closeness between replicate
concentrations of a sample and its mean value. It is expressed as the% relative
standard deviation about the mean (standard deviation/mean *100). Replicate (n
= 7) analysis of the samples were done on the same day (intra-day) and over a
period of two weeks (inter-day). Both intra-day and inter-day precision were
determined to be less than 10% for all the compounds. The intra-day and inter-
day precision values for codeine are shown in Table 3-2. Accuracy is expressed
as the difference between the actual value and the mean measured value for
each concentration. Accuracy determined from calibration graphs was less than
10%.
64
n
9>
IS
D.
350^
300..
250..
200..
150..
100..
50..
. Morphine i
.Codeine i
# ,,.| „ ,,|.i,.| MM |i m < m i | iiii|i m | m i|iiiil
50 100 150 200 250 300 350 400 450 500
Concentration (ng/ml)
Figure 3-5 : A typical calibration curve for codeine and morphine
in spiked urine.
9>
n
0)
a.
_»_M6G
_ci_M3G
_A-C6G
|..l.|llll|llll|llll|....|l H l|
50 100 150 200 250 300 350 400 450 500
Concentration (ng/ml)
Figure 3-6 : A typical calibration curve for codeine 6-glucuronide,
morphine 6-glucuronide and morphine 3-glucuronide in spiked urine.
m
3.1.6 stability
Stability of extracted plasma, urine and brain sannples were determined
over a 24 hour period by replicate analyses during the assay development and
validation. The variation in peak areas of samples determined from replicate
analysis was less than 10%.
Concentration
( ng/ml )
Intra-day variability
(%)
Inter-day variability
(%)
10
6.4 , ,
9.7
50
3.3
5.8
100
4.1
5.3
200
4.3
6.7
400
4.5
7.2
500
6.2
8.7
Table 3-2 : Intra-day and inter-day variability from replicate analysis of
standards containing various concentrations of codeine.
3.2 Synthesis
In the first step of the synthetic procedure, the key to the success of the
reaction was maintaining anhydrous conditions in the flask. This was achieved
by using a Dean-Starke trap, which allowed distillation of benzene and water at
regular intervals. The completion of the reaction was determined by the
disappearance of the starting material by thin layer chromatography. The
structure and identity of the compound formed after the first step was confirmed
66
1 ' . ■ .
by H NMR. It indicated the presence of acetyl and methyl groups and the
attachment of a glucuronic acid group to codeine. The melting point of the
compound was 113-116°C in agreement with the value reported by Yoshimura et
al. (1 968). The yield of this reaction was 70%.
Codeine 6-glucuronide was recrystallized from methanol and decomposed
at 225-230 °C, slightly lower than the value reported by Yoshimura et al. (1968).
This may be due to the fact that the product was apparently anhydrous
compared to the half-hydrate product reported in the reference paper. Absolute
methanol was used to recrystallize the compound instead of a water-methanol
mixture used in the reference. The identity of the product was confirmed by
comparing the retention time of the chromatographic peak to an analytical
standard by using the HPLC method previously described (section 2.1). The
overall yield of the reaction was about 16%.
3.3 Analgesia Studies
3.3.1 Intracerebroventricular Route
Intracerebroventricular administration of morphine, codeine, codeine 6-
glucuronide and the intermediate produced significant antinociceptive responses
in the rats. All the compounds tested produced a peak response about 20
minutes after administration. Each rat was also administered saline which
produced minimal changes to baseline responses. The data set for the effect of
surgery on the response time and the analgesic responses are summarized in
Tables 3-3 and 3-4, respectively. It was observed that the surgical procedure
67
itself had no effect on the response time (Figure 3-7) and the analgesic response
(Figure 3-8) and over a 3 hour period.
Tlme(mln)
1
2
3
4
5
Mean
SD
SEM
11.8
8.7
8.1
7.8
8.4
9.0
1.6
0.7
10
11.5
6.8
9.5
11.2
8.1
9.4
2.0
0.9
20
9.2
7.0
10.3
9.6
10.3
9.3
1.4
0.6
30
7.2
8.5
8.2
11.1
11.8
9.4
2.0
0.9
40
7.4
9.4
9.6
8.8
11.3
9.3
1.4
0.6
60
12.2
12.2
11.4
9.6
10.8
11.2
1.1
0.5
90
9.7
9.1
10.3
9.3
11.1
9.9
0.8
0.4
120
9.0
11.2
10.8
8.4
9.4
9.8
1.2
0.5
150
10.8
9.6
11.8
8.7
9.7
10.1
1.2
0.5
180
11.3
9.2
11.6
9.2
10.1
10.3
1.1
0.5
Table 3-3 : The data for the effect of intracerebroventricular
surgery on the response time (in seconds).
68
Time(min)
1
2
3
4
5
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
-1.1
-6.1
4.4
10.6
-0.9
1.4
6.3
2.8
20
-9.2
-5.4
6.9
5.6
6.0
0.8
7.5
3.4
30
-16.3
-0.6
0.3
10.2
10.8
0.9
11.0
4.9
40
-15.6
2.2
4.7
3.1
9.2
0.7
9.5
4.3
60
1.4
11.2
10.3
5.6
7.6
7.2
3.9
1.8
90
-7.4
1.3
6.9
4.7
8.5
2.8
6.3
2.8
120
-9.9
8.0
8.5
1.9
3.2
2.3
7.4
3.3
150
-3.5
2.9
11.6
2.8
4.1
3.6
5.4
2.4
180
-1.8
1.6
11.0
4.3
5.4
4.1
4.7
2.1
Table 3-4 : The data for the effect of intracerebroventricular
surgery on the analgesic responses (in% MPE).
69
40
^ 30
u
0)
42.
0)
.i 20
H
d
(A
o
0^ 10
.
.V
• I I I I^-^ ^ — I i i
"II ^
30 60 90 120 150 180
Time (min)
Figure 3-7 : The effect of the intracerebroventricular surgical procedure
on the response time.
100-
90^
*- 8o;.
u
"J 6o;.
1 ^°:-
X 40..
re
S 30..
**. 2o:.
10 !.
n
r r T T^^ 1 1 1
30 60 90 120 150 180
Time(min)
Fig
on
ure 3-8 : The effect of the intracerebroventricular surgical proc
the analgesic response.
edure
70
The response time (Tables 3-5 to 3-9) for all the treatments was
transformed into% of maximum effect data (Tables 3-10 to 3-14). The average
values from the data sets were then plotted as a function of time (Figures 3-9
and 3-10). Morphine was the most effective of the compounds, producing a
maximal suppression of pain, that is, 100% of the maximum possible effect
(MPE) in the rats. Codeine 6-glucuronide and the intermediate also exhibited
marked increases in antinociceptive responses, that is, up to 89 ± 6 and 81 + 9%
of MPE (± standard error of the mean or SEM), respectively. Codeine also
produced analgesia, with a peak effect of 63 ± 2% of MPE. The% MPE at peak
response time for all the compounds is summarized in Table 3-15. The area
under the effect curve (AUEC) was determined from individual% MPE versus
time graphs for each treatment using trapezoidal calculation (Figure 3-11) and is
summarized in (Table 3-16). < , , -,-<..'*.
3.3.2 Subcutaneous Route
Significant responses were seen with codeine, but not with codeine 6-
glucuronide or the intermediate after subcutaneous administration. There was
also a difference in the peak response time. While codeine produced a peak
response 30 minutes after administration, codeine 6-glucuronide and the
intermediate showed a peak response 45 minutes after they were administered.
The response time (Tables 3-17 to 3-20) for all the treatments was transformed
into% of maximum effect data (Tables 3-21 to 3-24). The average values from
the data sets were then plotted as a function of time (Figures 3-12 and 3-13).
Codeine exhibited a significant response, that is, 61 ± 5% of the MPE (Figure 3-
.i'N.'"-»-*i'7 '.•?'•
^,*.f;lfJ">l'"'M"W,
71
13). On the other hand, codeine 6-glucuronide and the intermediate produced
very poor responses, that is, 17 ± 3 and 1 1 + 2% of MPE, respectively. The%
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
10.7
10.4
10.4
13.3
11.9
13.1
11.6
1.3
0.5
10
28.1
18.9
18.8
30.6
26.3
18.6
23.6
5.4
2.2
20
33.3
40.0
38.1
40.0
38.9
31.6
37.0
3.6
1.5
30
29.2
40.0
33.3
40.0
37.2
29.7
34.9
4.9
2.0
40
24.8
40.0
28.3
36.6
31.8
23.6
30.9
6.5
2.7
60
22.6
33.7
24.2
32.7
27.7
21.7
27.1
5.2
2.1
90
15.2
27.6
22.7
30.5
26.9
18.6
23.6
5.8
2.4
120
13.3
24.4
20.9
26.9
24.8
18.0
21.4
5.1
2.1
150
13.4
20.1
19.7
17.7
19.9
17.2
18.0
2.6
1.0
180
12.1
11.3
16.8
15.1
12.6
16.6
14.1
2.4
1.0
Table 3-5 : The data for the effect of intracerebroventricular
administration of 10 ^g of codeine 6-glucuronide on the response time
(in seconds).
72
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
9.8
9.4
11.3
8.3
12.1
9.4
10.1
1.4
0.6
10
11.1
16.3
16.4
15.8
18.4
16.6
15.8
2.5
1.0
20
28.8
27.6
28.4
29.7
29.3
29.7
28.9
0.8
0.3
30
24.3
25.9
26.7
27.8
27.1
26.8
26.4
1.2
0.5
40
21.5
22.7
23.6
24.4
25.2
24.5
23.7
1.4
0.6
60
18.8
21.1
19.5
21.1
22.1
23.7
21.1
1.8
0.7
90
16.6
19.8
16.9
17.9
18.6
20.9
18.5
1.7
0.7
120
15.1
16.5
15.5
14.2
16.8
19.6
16.3
1.9
0.8
150
13.6
11.4
12.1
11.7
15.9
16.2
13.5
2.1
0.9
180
10.1
9.2
10.7
8.4
13.7
11.3
10.6
1.9
0.8
Table 3-6 : The data for the effect of intracerebroventricular
administration of 100 ng of codeine on the response time (in seconds).
73
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
14.4
11.4
14.2
10.0
12.9
12.7
12.6
1.7
0.7
10
40.0
14.6
22.3
33.1
19.7
22.6
25.4
9.4
3.8
20
40.0
28.1
40.0
40.0
28.1
32.8
34.8
5.9
2.4
30
40.0
24.4
36.4
40.0
27.3
30.7
33.1
6.7
2.7
40
28.3
19.3
28.8
40.0
21.0
24.6
27.0
7.4
3.0
60
18.1
19.8
24.9
40.0
20.3
20.4
23.9
8.2
3.3
90
18.6
15.3
21.7
29.3
17.7
17.8
20.1
5.0
2.0
120
14.7
11.1
19.2
27.8
16.9
16.3
17.7
5.6
2.3
150
13.6
10.7
18.6
22.7
15.8
14.7
16.0
4.2
1.7
180
12.8
9.8
16.4
14.6
13.2
12.3
13.2
2.2
0.9
Table 3-7 : The data for the effect of intracerebroventricular
administration of 10 |ig of the intermediate on the response time
(in seconds).
74
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
11.2
12.6
12.3
12.6
8.7
9.3
11.1
1.7
0.7
10
30.2
33.6
24.2
27.3
26.9
21.7
27.3
4.2
1.7
20
40.0
40.0
40.0
40.0
40.0
40.0
40.0
0.0
0.0
30
40.0
40.0
40.0
40.0
40.0
40.0
40.0
0.0
0.0
40
40.0
40.0
40.0
40.0
40.0
38.7
39.8
0.5
0.2
60
33.6
40.0
40.0
40.0
40.0
40.0
38.9
2.6
1.1
90
24.7
40.0
40.0
36.6
34.2
40.0
35.9
6.0
2.4
120
18.9
28.9
26.7
29.1
27.7
27.3
26.4
3.8
1.6
150
18.6
19.7
20.1
20.6
19.9
22.1
20.2
1.2
0.5
180
13.8
16.6
14.3
14.7
12.3
13.4
14.2
1.4
0.6
Table 3-8 : The data for the effect of intracerebroventricular
administration of 5 i^g of morphine on the response time (in seconds).
75
=====
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
11.2
10.4
10.3
11.0
11.4
8.7
10.5
1.0
0.4
10
10.6
11.7
11.2
12.8
13.1
9.9
11.6
1.2
0.5
20
12.1
12.3
12.3
13.2
13.6
10.2
12.3
1.2
0.5
30
14.4
11.6
14.3
12.6
12.9
10.9
12.8
1.4
0.6
40
15.2
10.9
14.8
13.9
13.9
11.3
13.3
1.8
0.7
60
14.1
11.1
12.5
14.1
12.6
10.6
12.5
1.5
0.6
90
12.9
10.8
13.8
12.7
13.1
9.7
12.2
1.6
0.6
120
11.4
11.2
14.1
13.6
13.6
11.3
12.5
1.4
0.6
150
11.0
11.6
12.2
12.1
13.4
11.5
12.0
0.8
0.3
180
10.5
10.5
9.8
11.2
13.3
10.8
11.0
1.2
0.5
Table 3-9 : The data for the effect of intracerebroventricular
administration of saline on the response time (in seconds).
76
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
4.3
22.5
17.8
23.7
22.6
23.5
19.1
7.6
3.1
20
62.9
59.5
59.6
67.5
61.6
66.3
62.9
3.4
1.4
30
48.0
53.9
53.7
61.5
53.8
56.9
54.6
4.4
1.8
40
38.7
43.5
42.9
50.8
47.0
49.3
45.4
4.5
1.8
60
29.8
38.2
28.6
40.4
35.8
46.7
36.6
6.8
2.8
90
22.5
34.0
19.5
30.3
23.3
37.6
27.9
7.2
2.9
120
17.5
23.2
14.6
18.6
16.8
33.3
20.7
6.8
2.8
150
12.6
6.5
2.8
10.7
13.6
22.2
11.4
6.7
2.7
180
1.0
-0.7
-2.1
0.3
5.7
6.2
1.8
3.4
1.4
Table 3-10 : The data for the effect of intracerebroventricular
administration of 100 jig of codeine on the analgesic responses
(in%MPE).
77
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
59.4
28.7
28.4
64.8
51.2
20.4
42.2
18.6
7.6
20
77.1
100.0
93.6
100.0
96.1
68.8
89.3
13.1
5.4
30
63.1
100.0
77.4
100.0
90.0
61.7
82.0
17.3
7.1
40
48.1
100.0
60.5
87.3
70.8
39.0
67.6
23.2
9.5
60
40.6
78.7
46.6
72.7
56.2
32.0
54.5
18.3
7.5
90
15.4
58.1
41.6
64.4
53.4
20.4
42.2
20.3
8.3
120
8.9
47.3
35.5
50.9
45.9
18.2
34.5
17.2
7.0
150
9.2
32.8
31.4
16.5
28.5
15.2
22.3
9.9
4.0
180
4.8
3.0
21.6
6.7
2.5
13.0
8.6
7.4
3.0
Table 3-1 1 : The data for the effect of intracerebroventricular
administration of 10 |ag of codeine 6-glucuronide on the analgesic
responses (in% MPE).
78
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
66.0
76.6
43.0
53.6
58.1
40.4
56.3
13.8
5.6
20
100.0
100.0
100.0
100.0
100.0
100.0
100.0
0.0
0.0
30
100.0
100.0
100.0
100.0
100.0
100.0
100.0
0.0
0.0
40
100.0
100.0
100.0
100.0
100.0
95.8
99.3
1.7
0.7
60
77.8
100.0
100.0
100.0
100.0
100.0
96.3
9.1
37
90
46.9
100.0
100.0
87.6
81.5
100.0
86.0
20.7
8.4
120
26.7
59.5
52.0
60.2
60.7
58.6
53.0
13.2
5.4
150
25.7
25.9
28.2
29.2
35.8
41.7
31.1
6.4
2.6
180
9.0
14.6
7.2
7.7
11.5
13.4
10.6
3.1
1.3
Table 3-12 : The data for the effect of intracerebroventricular
administration of 5 jig of morphine on the analgesic responses
(in% MPE).
79
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
100.0
11.2
31.4
77.0
25.1
36.3
46.8
34.1
13.9
20
100.0
58.4
100.0
100.0
56.1
73.6
81.4
21.3
8.7
30
100.0
45.5
86.0
100.0
53.1
65.9
75.1
23.7
9.7
40
54.3
27.6
56.6
100.0
29.9
43.6
52.0
26.4
10.8
60
14.5
29.4
41.5
100.0
27.3
28.2
40.1
30.6
12.5
90
16.4
13.6
29.1
64.3
17.7
18.7
26.6
19.2
7.8
120
1.2
-1.0
19.4
59.3
14.8
13.2
17.8
21.9
8.9
150
-3.1
-2.4
17.1
42.3
10.7
7.3
12.0
16.8
6.8
180
-6.3
-5.6
8.5
15.3
1.1
-1.5
1.9
8.5
3.5
Table 3-13 : The data for the effect of intracerebroventricular
administration of the 10 ng of the intermediate on the analgesic
responses (in% MPE).
Sv* ■■<
t v-y-*" - ■ ■^■<r-^ -^.r* -F^'
80
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
-2.1
4.4
3.0
6.2
5.9
3.8
3.6
3.0
1.2
20
3.1
6.4
6.7
7.6
7.7
4.8
6.1
1.8
0.7
30
11.1
4.1
13.5
5.5
5.2
7.0
7.7
3.7
1.5
40
13.9
1.7
15.2
10.0
8.7
8.3
9.6
4.8
2.0
60
10.1
2.4
7.4
10.7
4.2
6.1
6.8
3.3
1.3
90
5.9
1.4
11.8
5.9
5.9
3.2
5.7
3.5
1.4
120
0.7
2.7
12.8
9.0
7.7
8.3
6.9
4.4
1.8
150
-0.7
4.1
6.4
3.8
7.0
8.9
4.9
3.4
1.4
180
-2.4
0.3
-1.7
0.7
6.6
6.7
1.7
4.0
1.6
Table 3-14 : The data for the effect of intracerebroventricular
administration of saline on the analgesic responses (in% MPE).
81
40^
30
o
o
«
o
E
20
h-
m
Q.
0)
O
0^
10
■ ■ I ■
30
+-f
+
60
90
-.— I—
120 150
Time (min)
Cod
(100 meg)
.C6G
(10 meg)
Mor
(5 meg)
Int
(10 meg)
Saline
180
* (meg = micrograms). .V' -,
Figure 3-9 : Effect of Response Time to ICV Administration
of Saline, Codeine, Intermediate, Codeine 6-Giucuronide and Morphine.
Compound
% MPE
Peak Time
Morphine (5 |ig)
100 ±-
20 min
Codeine (100 |ig)
63 + 2
20 min
Codeine 6-glucuronide (10 [ig)
89 ± 6
20 min
Intermediate (10 i^g)
81 ± 9
20 min
Table 31-5 : % of maximum possible effect for various compounds at
the peak response time after i.c.v. administration.
82
o
£
UJ
E
3
E
X
(0
C6G
(10 meg)
Int
(10 meg)
Cod
(100 meg)
Saline
Mor
(5 meg)
30 60 90 120 150 180
Time (min)
* (meg = micrograms).
Figure 3-10 : Analgesic responses of rats to i.c.v. administration
of saline, codeine, intermediate, codeine 6-glucuronide and morphine.
Compound
Total AUECo.3h
Morphine (5 ^ig)
11 545 ±744
Codeine (100 i^g)
4462 ± 546
Codeine 6-glucuronide (10 i^g)
8156 ±1744
Intermediate (10 ^g)
6416 ±2602
Table 3-16 : Total AUECo.3h values after i.c.v. administration.
83
C6G Sal Cod Sal Int Sal Mor Sal
Figure 3-1 1 : The total area under the effect curve (AUEC0.3 h) after
i.c.v. administration.
The area under the effect curve (AUEC) was determined from individual% IVIPE
versus time graphs for each treatment using trapezoidal calculation (Figure 3-14)
and is summarized in Table 3-26.
3.3.3 Intravenous Route
Intravenous administration resulted in significant increases in the
responses associated with both codeine 6-glucuronide and the intermediate.
Codeine also produced a greater response by this route compared to the
subcutaneous route. As in the case of subcutaneous administration, there were
differences in the peak response times.
84
Time (min]
1
2
3
4
5
6
Mean
SD
SEM
10.9
9.9
11.6
8.7
10.8
12.2
10.7
1.2
0.5
15
18.2
16.3
17.4
18.3
20.5
22.3
18.8
2.2
0.9
30
26.9
27.7
28.9
27.3
29.9
30.2
28.5
1.4
0.6
45
21.3
25.1
26.3
26.6
28.7
28.6
26.1
2.7
1.1
60
20.6
24.4
24.7
25.9
28.3
27.8
25.3
2.8
1.1
90
18.7
22.2
23.6
25.4
27.9
27.7
24.3
3.5
1.4
120
17.6
20.1
21.1
23.5
25.8
25.4
22.3
3.2
1.3
180
16.8
18.7
20.3
21.7
22.6
23.3
20.6
2.5
1.0
240
14.9
16.6
17.5
20.2
20.6
21.1
18.5
2.5
1.0
300
13.8
14.8
15.2
18.3
17.7
18.4
16.4
2.0
0.8
360
12.3
12.2
13.7
15.1
13.2
13.8
13.4
1.1
0.4
Table 3-17 : The data for the effect of subcutaneous administration of
10 mg/kg of codeine on the response time (in seconds).
,--». -T y, 1-^ f-j-T'^'^'T"' 1 "? r"- ;•> " ■•»
85
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
8.8
10.6
10.3
9.4
13.1
13.2
10.6
1.8
0.6
15
12.3
12.1
12.5
12.2
11.3
10.9
11.9
1.6
0.5
30
14.7
14.8
12.8
13.6
12.1
12.2
13.5
2.3
0.8
45
18.2
17.3
14.7
13.9
12.7
13.7
15.3
3.6
1.2
60
16.8
16.5
12.9
13.7
14.6
13.9
14.9
2.9
1.0
90
16,7
14.9
12.6
12.9
13.3
12.6
13.9
2.9
1.0
120
13.0
14.5
13.5
12.7
13.1
12.2
13.2
1.8
0.6
180
11.4
13.7
11.7
11.2
12.7
12.3
11.8
1.6
0.5
240
9.8
12.1
11.2
11.9
11.9
11.9
11.3
1.2
0.4
300
10.4
11.3
11.8
10.8
11.3
11.8
11.3
0.7
0.2
360
8.9
11.0
11.9
10.3
10.8
11.2
10.6
1.1
0.4
Table 3-18 : The data for the effect of subcutaneous administration of
10 mg/kg of codeine 6-glucuronide on the response time (in seconds).
Time (min]
1
2
3
4
5
6
Mean
SD
SEM
9.5
9.9
10.1
10.2
12.1
10.1
10.0
1.0
0.3
15
9.8
11.5
10.6
10.4
11.3
9.5
10.3
0.8
0.3
30
10.2
13.3
10.6
12.3
10.9
11.2
11.3
1.1
0.4
45
12.5
13.8
11.3
13.8
12.6
11.6
13.0
1.3
0.5
60
13.2
13.7
12.5
13.9
13.1
12.8
13.2
0.7
0.2
90
13.4
12.6
12.9
12.9
13.3
12.7
13.0
1.2
0.4
120
12.7
12.7
11.7
12.6
12.8
11.9
12.6
1.1
0.4
180
11.9
13.2
12.3
11.8
12.2
11.3
12.1
0.6
0.2
240
12.6
12.5
12.6
11.9
11.7
10.8
11.9
1.1
0.4
300
12.7
11.9
10.7
11.1
11.6
10.7
11.4
0.8
0.3
360
12.0
10.7
9.6
11.4
10.3
10.2
10.8
1.0
0.3
Table 3-19 : The data for the effect of subcutaneous administration
of 10 mg/kg of the intermediate on the response time (in seconds).
87
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
8.1
8.5
8.6
9.8
9.6
10.8
9.8
1.4
0.5
15
9.8
10.0
8.1
10.7
9.3
9.2
9.8
0.9
0.3
30
10.1
10.8
10.2
10.2
10.0
9.9
10.3
0.5
0.2
45
11.0
10.2
11.3
10.1
10.1
11.2
10.9
0.7
0.3
60
9.8
11.1
9.8
10.0
9.9
11.7
10.6
0.8
0.3
90
8.9
10.8
9.8
10.2
10.2
12.2
10.6
1.0
0.4
120
8.6
9.3
10.7
10.3
10.3
11.9
10.3
1.0
0.4
180
10.3
8.9
11.3
9.5
10.7
11.7
10.5
0.9
0.3
240
10.6
9.4
10.7
9.7
9.8
10.6
10.1
0.5
0.2
300
11.2
8.6
11.3
9.9
9.9
10.4
9.9
1.0
0.4
360
11.1
9.7
10.6
10.1
10.4
10.2
10.2
0.6
0.2
Table 3-20 : The data for the effect of subcutaneous administration
of saline on the response time (in seconds).
> . f
88
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
15
25.1
21.3
20.4
30.7
33.2
36.3
27.8
6.6
2.7
30
55.0
59.1
60.9
59.4
65.4
64.7
60.8
3.9
1.6
45
35.7
50.5
51.8
57.2
61.3
59.0
52.6
9.2
3.8
60
33.3
48.2
46.1
55.0
59.9
56.1
49.8
9.6
3.9
90
26.8
40.9
42.3
53.4
58.6
55.8
46.3
12.0
4.9
120
23.0
33.9
33.5
47.3
51.4
47.5
39.4
11.0
4.5
180
20.3
29.2
30.6
41.5
40.4
39.9
33.7
8.4
3.4
240
13.7
22.3
20.8
36.7
33.6
32.0
26.5
8.9
3.6
300
10.0
16.3
12.7
30.7
23.6
22.3
19.3
7.7
3.1
360
4.8
7.6
7.4
20.4
8.2
5.8
9.0
5.7
2.3
Table 3-21 : The data for the effect of subcutaneous administration of
10 mg/kg of codeine on the analgesic responses (in% MPE).
89
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
15
4.5
5.1
10.2
7.4
2.8
9.2
6.9
3.0
1.0
30
11.6
14.3
21.6
8.4
5.9
13.7
12.8
6.1
2.0
45
28.4
22.8
28.3
14.8
2.5
14.7
16.6
9.8
3.3
60
27.1
20.1
21.9
8.8
4.7
14.1
13.9
7.3
2.4
90
18.8
14.6
21.2
7.7
0.6
11.4
11.1
7.7
2.6
120
9.6
13.3
16.3
10.8
5.6
10.8
11.3
3.9
1.3
180
4.1
10.5
5.7
4.7
1.6
5.9
5.7
3.2
1.1
240
0.0
5.1
3.5
3.0
3.7
8.2
4.7
2.1
0.7
300
1.0
2.4
2.8
5.1
8.1
4.6
4.6
2.3
0.8
360
2.7
1.4
-3.5
5.4
3.1
2.9
1.9
3.3
''
Table 3-22 : The data for the effect of subcutaneous administration of
10 mg/kg of the Intermediate on the analgesic responses (in% MPE).
'^n •i^-' - --^r^-
. -, *-■•
90
Time (min;
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
15
0.3
1.0
5.3
1.7
3.8
0.7
1.6
2.8
1.0
30
3.3
2.3
11.3
1.7
8.9
7.0
4.5
5.7
2.0
45
19.0
9.8
13.0
4.0
14.7
12.1
11.1
4.2
1.5
60
15.7
12.1
12.6
8.0
11.2
12.4
10.0
3.6
1.3
90
18.6
12.8
9.0
9.4
7.0
9.1
8.6
2.8
1.0
120
18.0
10.5
9.3
5.4
7.7
8.1
7.2
2.9
1.0
180
10.5
7.9
11.0
7.4
9.3
5.4
6.9
3.7
1.3
240
11.4
10.2
8.6
8.4
3.5
5.7
5.8
4.3
1.5
300
8.8
10.5
6.6
2.0
5.1
3.0
4.2
4.2
1.5
360
1
9.5
8.2
2.7
-1.7
4.8
4.0
1.9
5.2
1.8
Table 3-23 : The data for the effect of subcutaneous administration
of 10 mg/kg of codeine 6-glucuronide on the analgesic responses
(in% MPE).
91
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
15
11.3
11.6
13.1
13.9
8.3
0.7
8.0
6.5
2.5
30
15.6
17.5
16.9
20.8
16.2
-1.1
11.8
9.7
3.7
45
-2.8
13.4
2.1
20.2
17.8
-7.1
6.4
10.7
4.0
60
-1.1
14.4
7.6
21.5
15.0
-4.9
7.7
9.7
3.7
90
-4.3
9.1
3.8
18.6
12.7
-0.4
5.5
8.3
3.1
120
-1.1
7.2
10.3
17.0
13.1
-2.8
6.2
7.7
2.9
180
-5.0
11.6
-3.1
17.7
12.4
-1.8
4.7
9.0
3.4
240
-1.4
10.0
5.5
15.8
10.5
-3.2
5.1
7.4
2.8
300
-6.7
5.9
2.1
18.3
10.5
-7.4
2.8
9.5
3.6
360
-5.0
3.8
-7.9
12.6
9.9
-1.8
1.0
7.9
3.0
Table 3-24 : The data for the effect of subcutaneous administration
of saline on the analgesic responses (in% MPE).
92
o
o
E
H
CO
o
Saline
Int
10 mg/k
C6G
10 mg/k
Codeine
10 mg/k
I I 1 1 1 1 I I 1 1 1 1 1 1
180 240 300 360
Time (min)
Figure 3-12 : Effect of response time to s.q. administration
of saline, codeine, intermediate and codeine 6-glucuronide.
Compound
% MPE
Peak Time
Codeine (10 mg/kg)
61 ± 5
30 min
Codeine 6-glucuronide ( 10 mg/kg)
11 ± 2
45 min
Intermediate (10 mg/kg)
17 ± 3
45 min 11
Tgble 3-25 : % of maximum possible effect for various compounds at
the peak response time after s.q. administration.
93
o
UJ
E
3
E
X
S5
60 120 180 240
Time (min)
300
360
Figure 3-13 ; Analgesic responses to s.q. administration
of saline, codeine, intermediate and codeine 6-glucuronide.
1 Compound
Total AUECo.6h
Codeine (10 mg/kg)
10886 ± 646
Codeine 6-glucuronide (10 mg/kg)
1765 ± 159
Intermediate (10 mg/kg)
2321 ± 269
Table 3-26 : Total AUECo.6 h values after s.q. administration.
94
15000 ^
12000 . .
2 9000
Ul
<
CO 6000
o
3000 . .
ii
f
U
C6G Sal Cod Sal Int
Sal
Figure 3-14 : The total area under the effect curve (AUECo^h) after
s.q. administration.
The response time (Tables 3-27 to 3-30) for all the treatments was transformed
into% of maximum effect data (Tables 3-31 to 3-34). The average values from
the data sets were then plotted as a function of time (Figures 3-15 and 3-16).
Codeine exhibited a significant response, that is, 98 ± 4% of the MPE. Codeine
6-glucuronide and the intermediate produced responses which were 55 + 3 and
66 ± 3% of MPE, respectively. The% MPE at peak response time for all the
compounds is summarized in Table 3-35. The area under the effect curve
(AUEC) was determined from individual% MPE versus time graphs for each
treatment using trapezoidal calculation (Figure 3-17) and is summarized in Table
3-36.
95
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
8.7
11.8
9.8
12.8
10.1
11.4
11.1
1.6
0.6
10
27.6
31.3
33.7
31.7
29.8
27.7
29.3
3.4
1.3
20
38.4
37.9
36.9
40.0
39.1
37.2
38.5
1.2
0.5
30
35.9
34.1
33.6
37.7
36.3
34.5
35.7
3.1
1.2
40
32.7
33.8
32.9
34.3
35.4
32.8
33.3
3.6
1.4
60
23.8
22.2
26.9
26.4
30.6
25.1
25.7
2.7
1.0
90
19.7
18.1
22.7
23.1
24.3
23.7
21.7
2.3
0.9
120
18.1
17.7
20.3
20.3
21.0
18.8
19.0
1.6
0.6
180
14.8
15.2
19.7
19.7
19.9
16.3
17.2
2.5
0.9
240
13.2
14.4
16.8
16.6
9.4
13.2
13.8
2.5
0.9
300
11.6
12.3
14.7
14.5
10.0
12.7
12.6
1.6
0.6
Table 3-27 : The data for the effect of intravenous administration of
10 mg/kg of codeine on the response time (in seconds).
96
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
12.1
11.7
10.6
12.6
10.3
11.6
11.7
0.9
0.4
10
19.9
17
19.7
21.2
14.4
13.9
17.0
3.3
1.3
20
26.6
25.8
26.9
25.7
23.9
22.1
24.5
2.5
0.9
30
28.2
29.6
28.3
29.5
26.4
25.3
27.3
2.2
0.8
40
26.7
27.7
26.2
22.3
24.1
23.2
24.5
2.5
0.9
60
19.4
22.3
24.7
19.9
22.9
20.8
20.9
2.7
1.0
90
16.3
18.8
22.3
17.8
17.7
17.8
18.1
2.1
0.8
120
14.7
16.9
20.9
15.6
15.5
16.9
16.4
2.2
0.8
180
13.9
14.6
19.2
14.4
14.1
15.4
15.1
1.9
0.7
240
12.6
11.8
18.1
13.3
12.8
14.8
13.8
2.1
0.8
300
11.8
10.9
16.3
12.8
10.9
13.9
12.8
1.9
0.7
Table 3-28 : The data for the effect of intravenous administration of
10 mg/kg of codeine 6-glucuronide on the response time (in seconds).
97
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
10.1
11.6
9.1
12.2
11.2
13
11.3
1.3
0.5
10
17.6
20.5
15.7
19.1
17.5
18.2
17.9
1.6
0.6
20
23.9
24.8
24.2
24.5
23.6
25.1
24.4
0.5
0.2
30
32.7
33.9
30.7
29.1
28.7
29.6
30.4
2.2
0.8
40
27.9
26.5
25.1
25.2
26.0
27.7
25.8
2.0
0.8
60
25.4
19.0
23.2
23.9
22.3
22.3
22.4
2.1
0.8
90
22.4
17.6
18.1
21.2
20.8
21.1
20.0
1.8
0.7
120
18.8
16.5
16.4
18.4
17.7
19.7
18.0
1.2
0.5
180
14
15.6
14.8
17.1
17.3
17.8
16.3
1.5
0.6
240
13.7
14.8
12.7
16.2
16.6
16.9
15.1
1.6
0.6
300
12.8
13.1
11.1
15.5
14.7
14.6
13.5
1.5
0.6
Table 3-29 : The data for the effect of intravenous administration of
10 mg/kg of the intermediate on the response time (in seconds).
1 • « 'f i
98
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
9.3
9.9
11.1
10.1
13.0
12.2
11.2
1.5
0.6
10
10.8
10.3
10.7
10.4
12.7
12.8
11.5
1.2
0.5
20
10.6
10.6
11.0
11.1
11.8
13.1
11.6
1.0
0.4
30
11.3
10.8
11.9
10.8
13.5
12.7
12.0
1.1
0.4
40
11.0
11.1
12.3
10.7
12.2
12.2
11.9
1.0
0.4
60
10.9
11.2
11.6
11.1
13.3
12.8
12.1
1.2
0.5
90
11.3
11.2
11.7
10.0
12.7
12.9
11.8
1.1
0.4
120
11.6
10.8
12.2
11.5
12.2
12.3
11.9
0.7
0.3
180
10.9
10.9
11.9
11.3
13
12.6
11.9
0.9
0.3
240
11.1
10.6
11.3
10.6
13.1
13.6
11.9
1.3
0.5
300
10.6
10.9
11.9
9.8
13.2
13.2
11.7
1.3
0.5
Table 3-30 : The data for the effect of intravenous administration of
saline on the response time (in seconds).
99
Time (min)
1
2
3
4
5
6
MEAN
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
60.4
69.1
79.1
69.5
65.9
57.0
66.8
7.1
2.9
20
97.9
99.6
89.7
100.0
97.0
94.2
98.3
13.3
4.2
30
86.9
79.1
78.8
91.5
87.6
80.8
84.1
4.8
2.0
40
76.7
78.0
76.5
79.0
84.6
74.8
78.3
3.1
1.3
60
48.2
36.9
56.6
50.0
68.6
47.9
51.4
9.6
3.9
90
35.1
22.3
42.7
37.9
47.5
43.0
36.6
9.0
3.4
120
30.0
20.9
34.8
27.6
36.5
25.9
27.1
7.7
2.9
180
19.5
12.1
32.8
25.4
32.8
17.1
20.9
10.0
3.8
240
14.4
9.2
23.2
14.0
-2.3
6.3
9.5
8.6
3.3
300
9.3
1.8
16.2
6.3
-0.3
4.5
5.0
6.4
2.4
Table 3-31 : The data for the effect of intravenous administration of
10 mg/kg of codeine on the analgesic responses (in% MPE).
'(. »j/ - ' - -. >, -I .' f » 1
100
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
28.0
18.7
31.0
31.4
13.8
8.1
18.8
12.0
4.5
20
52.0
49.8
55.4
47.8
45.8
37.0
45.0
9.8
3.7
30
57.7
63.3
60.2
61.7
54.2
48.2
55.2
8.1
3.1
40
52.3
56.5
53.1
35.4
46.5
40.8
45.0
9.8
3.7
60
26.2
37.5
48.0
26.6
42.4
32.4
32.4
11.4
4.3
90
15.1
25.1
39.8
19.0
24.9
21.8
22.4
9.2
3.5
120
9.3
18.4
35.0
10.9
17.5
18.7
16.5
9.6
3.6
180
6.5
10.2
29.3
6.6
12.8
13.4
11.8
8.4
3.2
240
1.8
0.4
25.5
2.6
8.4
11.3
7.4
8.9
3.4
300
-1.1
-2.8
19.4
0.7
2.0
8.1
3.9
7.6
2.9
Table 3-32 : The data for the effect of intravenous administration of
10 mg/l<g of codeine 6-glucuronide on the analgesic responses
(in% MPE).
101
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
25.1
31.3
21.4
24.8
21.9
19.3
22.8
4.9
1.8
20
46.2
46.5
48.9
44.2
43.1
44.8
45.5
1.9
0.7
30
75.6
78.5
69.9
60.8
60.8
61.5
66.2
8.5
3.2
40
59.5
52.5
51.8
46.8
51.4
54.4
50.2
7.7
2.9
60
51.2
26.1
45.6
42.1
38.5
34.4
38.3
8.8
3.3
90
41.1
21.1
29.1
32.4
33.3
30.0
30.3
6.4
2.4
120
29.1
17.3
23.6
22.3
22.6
24.8
23.1
3.5
1.3
180
13.0
14.1
18.4
17.6
21.2
17.8
17.5
3.0
1.1
240
12.0
11.3
11.7
14.4
18.8
14.4
13.2
3.0
1.1
300
9.0
5.3
6.5
11.9
12.2
5.9
7.6
3.6
1.3
Table 3-33 : The data for the effect of intravenous administration of
10 mg/kg of the intermediate on the analgesic responses (in % MPE).
102
Time (min)
1
2
3
4
5
6
Mean
SD
SEM
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
10
4.9
1.3
-1.4
1.0
-1.1
2.2
0.9
2.2
0.8
20
4.2
2.3
-0.3
3.3
-4.4
3.2
1.0
3.1
1.2
30
6.5
3.0
2.8
2.3
1.9
1.8
2.8
1.8
0.7
40
5.5
4.0
4.2
2.0
-3.0
0.0
2.1
2.9
1.1
60
5.2
4.3
1.7
3.3
1.1
2.2
3.1
1.5
0.6
90
6.5
4.3
2.1
-0.3
-1.1
2.5
2.1
2.7
1.0
120
7.5
3.0
3.8
4.7
-3.0
0.4
2.3
3.5
1.3
180
5.2
3.3
2.8
4.0
0.0
1.4
2.2
2.3
0.9
240
5.9
2.3
0.7
1.7
0.4
5.0
2.2
2.4
0.9
300
4.2
3.3
2.8
-1.0
0.7
3.6
1.5
2.8
1.1
Table 3-34 : The data for the effect of intravenous administration of
saline on the analgesic responses (in% MPE).
103
u
o
E
I-
«
a
(0
&
Codeine
10 mg/kg
C6G
10 mg/kg
Int
10 mg/kg
Saline
I I I » I I I I I I I I I I I ■ I I
30 60 90 120 150 180 210 240 270 300
Time (min)
Figure 3-15 : Effect of response time to i.v. administration of saline,
codeine, intermediate and codeine 6-glucuronide.
1 Compound
% MPE
Peak Time
Codeine (10 mg/kg)
98 ± 4
20 min
Codeine 6-glucuronide ( 10 mg/kg)
55 ± 3
30 min
Intermediate (10 mg/kg)
66 ± 3
30 min
Table 3-35 : % of maximum possible effect for various compounds at
the peak response time after i.v. administration.
104
o
. 10 mg/kg
a_C6G
10 mg/kg
^_Int
10 mg/kg
^_ Saline
30 60 90 120 150 180 210 240 270 300
Time (min)
Figure 3-16 : Analgesic Responses to i.v. Administration of Saline,
Codeine, Intermediate and Codeine 6-Glucuronide.
1 Compound
Total AUECo.5h
Codeine (10 mg/kg)
8718 ±960
Codeine 6-glucuronide (10 mg/kg)
4781 ± 589
Intermediate (10 mg/kg)
5933 ± 289
Table 3-36 : Total AUECo-sh values after i.v. administration.
105
o
O
UJ
3
<
(0
o
15000 ^
12000 . .
9000 . .
6000..
3000 . .
C6G
Sal
Cod Sal
Int Sal
Figure 3-17 : The total area under the effect curve (AUEC0.5 n) after
i.v. administration.
3.4 Immune Studies
Codeine and its 6-glucuronide demonstrated inhibition of proliferation in
the PHA-stimulated lymphocytes, with maximal differences in cell inhibition of the
parent compound and its metabolite in the lower, physiologically relevant
concentration range of 0.156-5 jig/ml, as seen in Figure 3-18. Similarly, in the
case of the PMA-stimulated lymphocytes, there was very little suppression
observed at lower concentrations compared to the higher concentrations (Figure
3-19). Codeine and codeine 6-glucuronide also inhibited the MLR-stimulated
lymphocytes in a concentration-dependent fashion (Figure 3-20).
106
-20
156 313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-18 : The % inhibition of proliferation of PHA-stimulated T
lymphocytes after the addition of codeine and codeine 6-glucuronide.
* and * * Indicate a p-value of 0.05 and 0.01 , respectively.
313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-19 : The % inhibition of proliferation of PMA-stimulated T
lymphocytes after the addition of codeine and codeine 6-glucuronide.
* and * * indicate a p-value of 0.05 and 0.01 , respectively.
107
40
gCod
qC6G|
156
313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-20 : The% inhibition of proliferation of MLR-stimulated T
lymphocytes after the addition of codeine and codeine 6-glucuronide.
lymphocytes after the addition of codeine and codeine 6-glucuronide.Similar to
the effects of codeine and codeine 6-glucuronide, morphine and its 6-
glucuronide exhibited greatest inhibition of proliferation in the PHA-stimulated
cells (Figure 3-21). In the case of PMA-stimulated lymphocytes (Figure 3-22),
morphine and morphine 6-glucuronide demonstrated almost negligible inhibition
at lower concentrations (<1.25 fig/ml). These compounds also showed Inhibition
of the Mixed Lymphocyte Reaction (Figure 3-23).
When the intermediate was tested against codeine 6-glucuronide, it
showed a greater inhibition over the entire concentration range tested (0.156 to
10 ^g/ml) in all of the assays, that is, PHA (Figure 3-24), PMA (Figure 3-25) and
the MLR (Figure 3-26) models.
108
70
60
c
o
■^ 50
I
2 40
Q.
«*-
O
c 30
o
'■•3
£ 20
10..
|Mor
0M6G
156 313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-21 : The% inhibition of proliferation of PHA-stimulated T
lymphocytes after the addition of morphine and morphine 6-glucuronide.
* and * * indicate a p-value of 0.05 and 0.01 , respectively.
156 313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-22 : The% inhibition of proliferation of PMA-stimulated T
lymphocytes after the addition of morphine and morphine 6-glucuronide.
* and * * indicate a p-value of 0.05 and 0.01, respectively.
109
156 313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-23 : The% inhibition of proliferation of MLR-stimulated T
lymphocytes after the addition of morphine and morphine 6-glucuronide.
* and * * indicate a p-value of 0.05 and 0.01 , respectively.
60
|lnt
iC6G
156 313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-24 : The% inhibition of proliferation of PHA-stimulated T
lymphocytes after the addition of codeine 6-glucuronide and the
intermediate.
110
c
o
♦3
ig
I
o
a.
•3
c
o
*3
IS
IE
c
156 313 625 1250 2500 5000 10000
t Concentration (ng/ml)
Figure 3-25 : The% inhibition of proliferation of PMA-stimulated T
lymphocytes after the addition of codeine 6-glucuronide and the
intermediate.
40
35.
.2 30..
2
(b
S 25 4-
o
Q.
•S 204-
c
o
9
15..
£ 10..
5i
□
Int
C8G
Jl
1-,
156 313 625 1250 2500 5000 10000
Concentration (ng/ml)
Figure 3-26 : The% inhibition of proliferation of MLR-stimulated T
lymphocytes after the addition of codeine 6-glucuronide and the
intermediate.
T T"^ IT," r ■« -
111
3.5 Receptor Binding Studies
Morphine exhibited the highest affinity in the receptor binding assays, with
an IC50 value of 1.8 ± 0.6 nM (Table 3-9). In the case of codeine, however, the
binding curve was shifted significantly to the right of the concentration range
(IC50 = 1.5 ± 0.4 |iM ) indicating a much weaker affinity for the |i receptor.
Codeine 6-glucuronide had a slightly lower binding compared to codeine (IC50 =
4.4 ± 0.2 fxM). The intermediate, on the other hand, exhibited a slightly higher ^
receptor binding affinity compared to codeine and codeine 6-glucuronide (IC50 =
0.13 ± 0.06 ^M).
The raw counts per minute (CPM) data of each compound were converted
into the more standardized form, that is, % of total binding. The receptor binding
profiles of morphine (Figures 3-27 and 3-28), codeine (Figures 3-29 and 3-30),
codeine 6-glucuronide (Figures 3-31 and 3-32) and the intermediate (Figures 3-
33 and 3-34) are represented in the graphs below.
Parameters
Mor
Cod
C6G
Int
T (cpm)
3358
±343
2609
±350
2249
±107
2479
±481
NS (cpm)
961
±97
876
±24
706
±64
779
±141
N
0.98
±0.42
0.96
±0.26
1.02
±0.28
1.13
±0.36
IC50
1.81
±0.58
nM
1.49
±0.39
^M
4.4
±0.23
HM
0.13
±0.06
l^M
Table 3-37 : The |i receptor binding parameters for morphine, codeine,
codeine 6-glucuronide and the intermediate.
112
3.500
2.900
2.300
1.700 -
1.100
0.500
10-12 10" 10-i« 10-» 10-* 10-' 10-*
Concentration (M)
Figure 3-27 : The receptor binding profile of morphine.
(CPM vs concentration).
105
1
S
1
' ' ' I '— ^-n ' — ' ' I ' — n "— "-^
10-12 -I0-" 10" 10-» 10* 10-' io-»
Concentration(M)
Figure 3-28 : The receptor binding profile of morphine.
(% total binding vs concentration).
flL
u
2.600
2.267
1.933
? 1.600
1.267
0.933 -
0.600
10'
113
Concentration (M)
■^^T 1 TT-i 1 r-T^ 1 ^f] 1 — n-1 , r ,
10-* 10-' 10-* 10* 10-* 10-3
Figure 3-29 : The receptor binding profile of codeine.
(CPM vs concentration).
1
S
I
10-« 10-*
10-7 10-« 10-5
Concentration(M)
10^
10-
Figure 3-30 : The receptor binding profile of codeine.
(% total binding vs concentration).
114
u
z.u -
*-.
S^
1.8-
X
1.6-
• " ■
\
14-
\
r *^
1.2-
■ 5 -
■■'■: :
...3
'I ^
1.0-
y
;A /■■
\(''.' r. '
.
■' "■-
'.,■
0.8-
' ' 'I'
1 n <—
— •
0.6-
•
i-r-| ,—r-r
10-9 10-« 10' io-« 10-*
Concentration (M)
io-»
10-s
Figure 3-31 : The receptor binding profile of codeine 6-
glucuronide. (CPI\/1 vs concentration).
1
I
10^ 10-* 10-' 1G-« 10*
Concent ration(M)
10-* 10-3
Figure 3-32 : The receptor binding profile of codeine 6-
glucuronide. (% total binding vs concentration).
115
e
8
M
S
a.
u
2.500
2.100 -
1.700
1.300 -
0.900
0.500
10^ 10-»
10'
Concentration (M)
-q ^^^T ^^-q , r-
10-" 10* 10-* 10-3
Figure 3-33 : The receptor binding profile of the intermediate.
(CPM vs concentration).
at
e
e
s
I
io-« io-« 10-' io-« 10-5
Concentration (IM)
10-«
10-3
Figure 3-34 : The receptor binding profile of the intermediate.
(% total binding vs concentration).
116
3.6 Plasma and Brain Concentrations
System 2 was used to determine concentrations of codeine, morphine and
their glucuronides in rat plasma and brain samples. Extractions were done using
disposable Clean Screen® columns as in the case of blank human urine used to
develop the HPLC method. However, for the rat plasma and brain samples, a
10% ammonium hydroxide solution in methanol was required to obtain good
recoveries of the polar glucuronide metabolites. The recoveries were better than
80% for all the compounds.
Intracerebroventricular administration of codeine (Figure 3-35) in rats
resulted in the partial conversion of codeine to morphine in the brain (Table 3-
38). However, no drug was detected in the plasma. When codeine 6-glucuronide
was administered directly into the brain, a small amount of morphine 6-
glucuronide was detected in the brain (Table 3-39) but no drug was detected in
the plasma.
After subcutaneous administration of codeine, it was seen that some
codeine was converted into morphine and morphine 3-glucuronide in the plasma.
Neither morphine 6-glucuronide or codeine 6-glucuronide were detected in the
plasma. However, both morphine 6-glucuronide and codeine 6-glucuronide were
present in small quantities in the brain of some of the rats. After subcutaneous
administration of codeine 6-glucuronide, no drug or its metabolites was detected
either in the plasma or the brain. Therefore, this route of administration was not
pursued.
After intravenous administration of codeine (Figure 3-36), some of the
codeine was converted into morphine, morphine 3-glucuronide and codeine 6-
glucuronide were found in the plasma (Table 3-40). In the brain, small amounts
117
of both morphine 6-glucuronicle and codeine 6-glucuronide were detected (Table
3-41). Codeine 6-glucuronide administration resulted in the presence of only
codeine 6-glucuronide in the plasma (Table 3-42). However, in the brain, small
amounts of both codeine 6-glucuronide and morphine 6-glucuronide were
detected (Table 3-43). The results obtained after administration of codeine and
codeine 6-glucuronide by the various routes are summarized in Table 3-44.
Sample
number
Codeine
(ng/g)
Morphine
(ng/g)
1
9652
1803
2
7965
1046
3
8242
1287
4
7191
1311
5
6647
1207
6
8018
1379
Mean ± SD
7953 + 1026
1339 + 254
Table 3-38 : Concentrations in the rat brain after i.c.v. administration
of 100 |ig codeine.
. — 1 ^
Sample
number
C6G
(ng/g)
M6G
(ng/g)
1
815
63
2
744
43
3
917
57
4
1033
78
5
668
53
6
791
69
Mean + SD
828 + 130
61 +12
Table 3-39 : Concentrations in the rat brain after i.c.v. administration of
10 fig codeine 6-glucuronide.
118
A-
12.00 i4!oo is.oo i»;od 2o!od ii'.oo ' zi'.to 'le'.oo jg
Figure 3-35 : Chromatogram of a brain sample after administration of 100
fig of codeine i.c.v. in the rat.
0.0«-
0.05-
2
0.04-
A
0.03-
0.02-
M
S
t»' ' '-
, '■'"-- •■•''
O.Ol-
J
V.
8
U
0.00-
r — ^ ---^
12
00
; r— T :
14.00
18.00 U.OO
20.00
22.00
24:00 '2«
Figure 3-36 : Chromatogram of a plasma sample after administration
of 10 mg/kg codeine i.v. in the rat.
119
Sample
number
Codeine
(ng/ml)
Morphine
(ng/ml)
C6G
(ng/ml)
M3G
(ng/ml)
1
2187
199
28
469
2
3352
239
36
564
3
2678
178
ND
447
4
1787
113
ND
239
5
1886
146
ND
348
6
2013
167
21
457
Mean ± SD
2313 + 596
174 + 43
28 + 8
421 ±112
Table 3-40 : Concentrations in the rat plasma after i.v. administration of
1 mg/kg of codeine.
1 Sample
number
Codeine
(ng/g)
Morphine
(ng/g)
C6G
(ng/g)
M6G
(ng/g)
1
247
51
27
21
2
319
49
34
25
3
276
42
23
ND
4
231
37
21
ND
5
198
29
ND
ND
6
257
45
ND
18
Mean + SD
255 + 41
42 + 8
26 + 6
21 ±4
Table 3-41 : Concentrations in the rat brain after i.v. administration
of 1 mg/kg of codeine.
Sample
number
C6G
(ng/ml)
1
4736
2
3773
3
4062
4
5168
5
4183
6
3137
Mean + SD
4177 + 715
Table 3-42 : Concentrations in the rat plasma after i.v. administration
of 10 mg/kg codeine 6-glucuronide.
120
Sample
number
C6G
(ng/g)
M6G
(ng/g)
1
197
29
2
161
ND
3
258
27
4
396
41
5
142
ND
6
218
35
Mean ± SD
229 ± 92
33 ±6
Table 3-43 : Concentrations in the rat brain after i.v. administration
of 10 mg/kg codeine 6-glucuronide.
Drug Administration
Plasma
Brain
Codeine - ICV
-
CM
C6G - ICV
-
C6G, M6G
Codeine - SQ
C, M, M3G
C, M, C6G, M6G
C6G - SQ
-
-
Codeine - IV
C, M, C6G, M3G
C, M, C6G, M6G
C6G - IV
C6G
C6G, M6G II
Table 3-44 : Qualitative summary of results after administration of
codeine and codeine 6-glucuronide by various routes.
.■'*> ■y
CHAPTER 4
DISCUSSION
The overall aim of this research project was to examine and compare the
analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide.
To achieve this objective the following hypotheses were formulated.
1. Codeine 6-glucuronide, like morphine 6-glucuronide, possesses
analgesic activity.
2. The glucuronide metabolites of codeine and morphine are less
immunosuppressive than their parent compounds.
The rat was found to be a good model for the analgesia studies since
negligible amounts of codeine 6-glucuronide are present in the rat plasma and
urine after codeine administration. This allowed direct comparison of the
antinociceptive activities of codeine 6-glucuronide with codeine without the
contribution of codeine 6-glucuronide to the analgesia of codeine.
An HPLC-UV based method was successfully developed for the analysis
of codeine, morphine, codeine 6-glucuronide, morphine 6-glucuronide and
morphine 3- glucuronide in biological samples, that is, human urine, rat plasma
and rat brain. An efficient solid phase extraction method was used to obtain
clean extracts and excellent recoveries in excess of 80% for all the compounds
of interest. Calibration curves for each drug were linear over the concentration
range 0-500 ng/ml in all the biological samples with a correlation coefficient of
0.99 or better.
121
122
During the synthetic procedure, an intermediate compound formed after
the first reaction step was isolated and characterized. This intermediate
represents the glucuronic acid moiety attached to the 6-position of codeine with
intact acetyl/methyl groups. It appears to be comparatively more lipophilic than
codeine 6-glucuronide and should be capable of crossing the BBB more easily.
In these studies the exhibited antinociceptive activity which was similar to that of
codeine 6-glucuronide itself. This indicates that this kind of "prodrug" might be a
viable approach for delivering the potent glucuronides across the BBB.
Intracerebroventricular administrations of morphine, codeine, codeine 6-
glucuronide and the intermediate resulted in significant analgesic responses. All
the compounds exhibited greater areas under the effect curve (AUECs)
compared to saline treatment (p<0.01). Morphine (dose = 5 ^g) AUEC value was
seen to be statistically higher than codeine 6-glucuronide (dose = 10 ^g),
intermediate (dose = 10 |ag) and codeine (dose = 100 ^g), with p< 0.01. Both
codeine 6-glucuronide and the intermediate exhibited larger AUECs compared to
codeine (p<0.05). However, there were no statistically significant differences
between codeine 6-glucuronide and the intermediate. Using this method, the
rank order of effectiveness based on the AUEC values was morphine > codeine
6-glucuronide z intermediate > codeine > saline.
The analgesic effects of codeine 6-glucuronide and the intermediate were
not statistically different from saline treatment after the subcutaneous studies.
This lack of activity is probably associated with the limited ability of the polar
glucuronides to cross the BBB and the poor absorption of these hydrophilic
molecules from the subcutaneous tissue into the systemic circulation. Codeine
(dose = 10 mg/kg) exhibited a statistically significant AUEC (p<0.01) compared
123
to codeine 6-glucuronide (dose = 10 mg/kg), intermediate (dose = 10 mg/kg) and
saline treatments. There were no significant differences among the other
treatments, that is, codeine 6-glucuronide, intermediate and saline. Comparison
of total AUEC values indicated that at the dose administered, the order of
effectiveness was codeine » codeine 6 -glucuronide z intermediate r saline.
The intravenous route was chosen in order to remove one of the rate-
limiting factors, that is, absorption. There was a significant increase in the
responses associated with both codeine 6-glucuronide (dose = 10 mg/kg) and
the intermediate (dose = 10 mg/kg). Codeine (dose = 10 mg/kg) also produced a
greater response by this route compared to the subcutaneous route. This
indicates that absorption of these compounds from the subcutaneous tissue can
represent a significant barrier to the pharmacodynamic responses. All of the
compounds exhibited greater areas under the effect curve compared to saline
treatment (p<0.01). The codeine AUEC value was higher than codeine 6-
glucuronide and the intermediate (p<0.01). However, there were no significant
differences between the AUECs of codeine 6-glucuronide and the intermediate.
Comparison of total AUEC values indicate that at the dose administered, the
order of effectiveness was codeine > codeine 6 -glucuronide ~ intermediate>
saline.
All four agents tested, that is, codeine, morphine, morphine 6-glucuronide
and codeine 6-glucuronide, demonstrated an immunomodulatory effect in all of
the three different in vitro systems. Cell viability was greater than 98% in all
cases as determined by the tryphan blue staining method. The differences
between the effects of codeine and its 6-glucuronide in the PHA assay were
more apparent at lower concentrations, that is, 0.156 to 0.625 jig/ml (p<0.01).
124
This data suggests that both codeine and codeine 6-glucuronide inhibit the T
cell responses via the first signal activation pathway, which is mediated through
interleukin-2 and calcium channel pathways.
In the case of PMA-stimulated lymphocytes, the 6-glucuronide showed no
inhibition in the lower, physiologically relevant concentrations (0.156 and 0.313
|ig/ml) compared to codeine. The 6-glucuronide demonstrated significantly less
inhibition than codeine in all concentrations tested (p< 0.05). Codeine and
codeine 6-glucuronide also inhibited the MLR-stimulated lymphocytes in a dose-
dependent fashion. However, there was no statistical difference between the
effects of the two agents.
Morphine and its 6-glucuronide also inhibited the PHA-stimulated cells in
a concentration-dependent manner. Morphine 6-glucuronide, like codeine 6-
glucuronide, produced almost no inhibition in the lower concentration range. In
the concentration range of 0.156-2.5 iig/ml, the inhibitory effects of the
glucuronide metabolite were less compared to its parent compound (p<0.05). As
in the case of codeine and codeine 6-glucuronide, the differences were more
apparent in the lower concentrations, that is, 0.156 to 0.625 ^g/ml (p<0.01).
In the case of PMA-stimulated lymphocytes, morphine and morphine 6-
glucuronide demonstrated almost negligible inhibition at lower concentrations
(<1.25 ^g/ml).. At higher concentrations, however, both compounds showed a
marked inhibition of proliferation. These compounds also showed inhibition of the
mixed lymphocyte reaction (MLR). The 6-glucuronide of morphine showed less
immunosuppression compared to morphine over the entire concentration range
(0.156-10 |ig/ml). The decreased immunosuppressive effects of morphine 6-
125
glucuronide in comparison to morphine were statistically significant at
concentrations below 0.625 |ig/ml (p<0.05).
Both morphine and codeine exert their effects by interacting with the ^
opioid receptors in the central nervous system. The stronger analgesic activity of
morphine compared to codeine is usually attributed to its high receptor binding
affinity. In this study, the affinity profiles of morphine, codeine, codeine 6-
glucuronide and the intermediate towards the ^ opioid receptor were examined.
It was seen that morphine exhibited the highest affinity, with an IC50 value of 1.8
± 0.6. In the case of codeine, however, the binding curve was shifted significantly
to the right of the concentration range (IC50 = 1.5 + 0.4 jaM), indicating a much
weaker affinity for the n receptor. This is in agreement with reports in the
literature, which suggest a 1000 to 3000 fold less affinity for codeine compared
to morphine (Chen et al., 1990 ; Mignat et al., 1995). Codeine 6-glucuronide
exhibited slightly lower binding compared to codeine (IC50 = 4.4 + 0.2 |iM). This
indicated that glucuronidation of codeine did not change the receptor affinity
significantly. The intermediate, on the other hand, exhibited a higher ^ receptor
binding compared to codeine and codeine 6-glucuronide (IC50 = 0.13 ± 0.06 ^M).
This enhanced binding might be due to the fact that the intermediate is more
lipophilic in nature than codeine and codeine 6-glucuronide.
After intracerebroventricular administration of codeine, some of the
codeine was converted into morphine in the brain. No codeine 6-glucuronide or
any morphine glucuronides were detected. There was no drug detected in the
plasma. When codeine 6-glucuronide was administered, there was a partial
formation of morphine 6-glucuronide from codeine 6-glucuronide. As in the case
of codeine administration, no drug was detected in the plasma.
126
After subcutaneous administration of codeine, it was seen that some of
the codeine was converted into morphine in the plasma. Morphine 3-glucuronlde
was also detected in the plasma, but no morphine 6-glucuronide or codeine 6-
glucuronide were detected. In the brain, however, both morphine 6-glucuronide
and codeine 6-glucuronide were detected. This indicates that the glucuronides
can cross from the blood to the brain. In the case of subcutaneous administration
of codeine 6-glucuronide, however, no drugs could be detected either in the
plasma or the brain. This is most likely due to the very poor absorption of
codeine 6-glucuronide from the subcutaneous tissue.
After intravenous administration of codeine, some codeine was converted
into morphine. Morphine 3-glucuronide was also detected in the plasma, but not
morphine 6-glucuronide. There was also a small amount of codeine 6-
glucuronide detected in the plasma. In the brain, both morphine 6-glucuronide
and codeine 6-glucuronide were present. Codeine 6-glucuronide administration
resulted in the presence of only codeine 6-glucuronide in the plasma, showing
that it is stable in the plasma and does not convert to either codeine, morphine or
morphine 6-glucuronide. In the brain, some codeine 6-glucuronide was seen to
be 0-demethylated to morphine 6-glucuronide. Therefore, the analgesic
responses observed in the rats would appear to be associated with codeine 6-
glucuronide and to some extent, morphine 6-glucuronide.
Codeine, although a chemical congener of morphine, is more widely used
as an analgesic since it has substantially less potential for the development of
dependence. It has been suggested that the analgesic action of codeine is
associated with its conversion to morphine in the body. If this was the case
however, one would expect to see relatively high concentrations of morphine in
127
the plasma and urine after codeine administration. However, only small amounts
of morphine were detected after oral administration of codeine by various
investigators (Quiding et al., 1986 ; Shah and Mason, 1990 a ; Persson et al.,
1992). In addition, urinary excretion of morphine has been reported to be only
0.2-0.8% of the total codeine dose (Yue et al, 1990 a, b ; Chen et al., 1991). This
is consistent with the report of Persson et al. (1992) that the ratio of the AUG of
morphine to codeine is only 3%. Sindrup et al. (1990) found morphine
concentrations of up to 13 ng/ml in extensive metabolizers 90 minutes after oral
administration of 75 mg of codeine. In contrast, Findlay et al. (1986) and Guay et
al. (1987) have reported concentrations of morphine up to 16 ng/ml after an oral
dose of 60 mg of codeine, which theoretically could contribute to the analgesic
effect of codeine.
Although these reports regarding morphine levels after codeine treatment
are contradictory, it is important to recognize that high morphine concentrations
were all obtained using radioimmunoassays for morphine (Findlay et al., 1986 ;
Guay et al., 1987), while other studies in which low or undetectable
concentrations were reported used either HPLC (Chen et al., 1991 ; Shah and
Mason, 1991 ; Yue et al., 1990 a, b) or GC-MS (Quiding et al., 1986) techniques.
The apparent high concentrations of morphine may therefore be associated with
cross-reactivity of the antibody to other opiates. Thus, in view of the low
morphine concentrations it is most likely that its contribution to the analgesic
effect of codeine is small. However, it has been hypothesized that O-
demethylation to morphine may take place in the vicinity of receptors in the CNS
(Chen et al., 1990). It has also been suggested that codeine may act on different
128
opioid receptors (Neil, 1984) and, therefore, may itself mediate a large part of its
analgesic effect.
There has been no documented study regarding the activity of codeine 6-
glucuronide, the major metabolite in some species including humans. In contrast
to the low concentrations of morphine produced after codeine administration in
humans, concentrations of codeine 6-glucuronide have been reported to be 10-
1 5 times those of codeine itself. We have shown that not only does codeine 6-
glucuronide cross the blood brain barrier, it also has potent analgesic activity.
This is in agreement with previous work describing the activity of morphine 6-
glucuronide.
Yoshimura et al. (1973) reported that both morphine 6-glucuronide and
morphine 3-glucuronide could cross the blood brain barrier and act on the opioid
receptors in the brain of rats. Other animal studies done in both rat and mouse
have shown that morphine 6-glucuronide has a higher affinity for p opioid
receptors than morphine itself (Abbott and Palmour, 1988 ; Pasternak et al.,
1980 ; Paul et al., 1989 ; Gong et al., 1991) and is 2-4 times more potent as an
analgesic than morphine when injected subcutaneously as assessed by
antinociceptive tests, that is, tail flick and hot plate tests (Gong et al., 1992 ;
Hannaetal., 1990).
Morphine 3-glucuronide, on the other hand, was found to have a very low
affinity for |j opioid receptors in the brain and did not exhibit any analgesic effect
when administered in pain-induced rat and mouse. Direct clinical evidence for
the analgesic activity of morphine 6-glucuronide has been reported in man
(Osborne et al., 1988 , 1990). It was seen that lower doses of morphine 6-
glucuronide were needed compared to morphine for the same extent of pain
129
relief in cancer patients (Hanna et a!., 1990 ; 1991). Our results show that like
morphine 6-glucuronide, codeine 6-glucuronide possesses analgesic activity of
its own. . 'y ■> ■
The concept of opiates as imnnunomodulatory agents has long been
recognized. It is well known that cell-mediated immune function Is impaired in
heroin and opiate addicts (Brown et al., 1974) and that the major cell types
involved in cell-mediated immunity, that is, T lymphocytes and mononuclear
phagocytes, possess opiate receptors (Wybran et al., 1979 ; McDonough et a!.,
1980 ; Lopker et al., 1980). Opiate addicts have been shown to exhibit an
increased incidence of bacterial, protozoal, fungal and viral infections (Bryant et
al., 1990). Until recently, this was attributed to the sharing of unsterilized and
contaminated needles by the addicts. However, there is now a considerable
body of evidence suggesting that opiates themselves cause suppression of
various immunological endpoints.
The immunosuppressive effects of opiates may have potentially important
clinical relevance, particularly in the case of individuals having a high degree of
susceptibility towards infectious diseases. Effects on immunocompetence is a
major area of concern when dealing with organ transplants, burns, cancer,
rheumatoid arthritis and other autoimmune diseases.
The relationship between the receptor binding affinity of codeine 6-
glucuronide and its in vivo potency seems to be inconsistent. The glucuronide is
analgesically much more active than its parent compound, but its receptor affinity
does not reflect this observation. There are a few possible explanations. First,
binding studies performed with brain homogenates do not necessarily represent
the true physiological conditions. At best they can give a good approximation
130
regarding the activity of the compounds. They only indicate the binding of
compounds to receptors under the specified conditions of the assay. However,
this does not accurately estimate the efficacy of the compounds in vivo in
producing a physiological response. Second, differences in the distribution,
metabolism and elimination of these compounds can play a major role in
modulating the pharmacodynamic responses. This too cannot be taken into
account in binding studies. Third, these compounds may possess higher affinity
towards other opioid (delta, kappa etc.) or even non-opioid receptors and this
interaction may account for the enhanced analgesic potency of codeine 6-
glucuronide in vivo. The lesser affinity of the glucuronide for the ^ receptor may
also account for its lower immunosuppressive effects.
Shashoua et al. (1986) have identified UDP-glucuronlc acid, a co-
substrate for the glucuronidation reaction, in the rodent brain. There is also
evidence of the presence of glucuronosyl transferase enzymes in the brain of
rodents and humans (Wahlstrom et al., 1988). This raises the possibility that
glucuronidation occurs in the brain. At present this seems unlikely and further
investigation needs to be done in order to validate this hypothesis. It is more
reasonable to assume that codeine 6-glucuronide produced in the liver is able to
cross the blood brain barrier by an, as yet, unknown mechanism.
The unexpected behavior of the hydrophilic conjugates of codeine and
morphine with regards to crossing the blood brain barrier was partly explained by
Carrupt et al. (1991). It was suggested that glucuronides existed in a
conformational equilibrium between their hydrophilic and lipophilic forms,
depending on the surrounding media. In the vicinity of the BBB the glucuronides
were seen to be present in their lipophilic form which allowed easier penetration
131
into the brain. As the glucuronides circulating in the blood were in much higher
concentration compared to their parent compounds, this caused a sufficient
concentration gradient to be established, so as to allow the glucuronides to
passively diffuse across the blood brain barrier (Barjavel et al., 1995).
In conclusion, the data presented in this thesis suggests that the
glucuronide metabolites of codeine and morphine may be more beneficial in the
treatment of patients with low immunocompetence experiencing clinical pain
during trauma and even after major surgery. Hence, both codeine 6-glucuronide
and morphine 6-glucuronide, along with related compounds like the intermediate,
deserve further assessment as potential agents for clinical pain management in
the case of patients with a compromised immune function.
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BIOGRAPHICAL SKETCH
Vinayak Jaya Srinivasan was born on the 24th of April, 1969, in Kolar,
India. He obtained his bachelors degree in pharmacy from the Institute of
Technology - Banaras Hindu University in May, 1991. He started graduate
studies in the College of Pharmacy at the University of Florida in August, 1991.
After graduation, he plans to pursue a career in pharmaceutical research.
' : , i ^- '. " i ' ' ' ^ '.
ij
149
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
"^ ^T^.v5^
Ian R. Tebbett, Chair
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Donna Wielbo
Assistant Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
U 01)^^4-
Hartmut Derendorf
Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philososhy.
Kenneth Sloan
Professor of Medicinal Chemistry
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentatkm^nd Is fully adeqtratevNin scope
and quality, as a dissertation for the degree of Doctor of Ph]losophy^
(j><~<K.M^ I - ' ^4-/l..-V--c4-t>v--X
Roger Bertholf
Associate Professor of Pathology
and Laboratory Medicine
This dissertation was submitted to the Graduate Faculty of the College of
Pharmacy and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy. /
May, 1996
Dean, College of Pharmacy
Dean, Graduate School
UNIVERSITY OF FLORIDA
3 1262 08555 3195
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